Compounds and methods for detection of carcinomas and their precursor lesions

ABSTRACT

The present invention relates to compounds and methods for detection and treatment of carcinomas and their precursor lesions. The invention provides DNase nucleic acids and polypeptides useful for the detection and treatment of carcinomas and their precursor lesions. The invention is more specifically related to a method for detection of carcinomas and their precursor lesions comprising the detection of the level and/or the subcellular localization of one or more DNase molecules in biological samples. Furthermore the present invention provides methods for early diagnosis, prognosis and monitoring of the disease course of carcinomas and their precursor lesions as well as for the treatment of said lesions.

The present invention relates to compounds and methods for detection andtreatment of carcinomas and their precursor lesions. The inventionprovides DNase nucleic acids and polypeptides useful for the detectionand treatment of carcinomas and their precursor lesions. The inventionis more specifically related to a method for detection of carcinomas andtheir precursor lesions comprising the detection of the level and/or thesubcellular localization of one or more DNase molecules in biologicalsamples. Furthermore the present invention provides methods for earlydiagnosis, prognosis and monitoring of the disease course of carcinomasand their precursor lesions as well as for the treatment of saidlesions.

In most tumours there is a strong correlation between the patientsoutcome following initial therapy and the stage at which the disease hasbeen diagnosed. So the earlier the cancer could be detected the betterare the chances for the patient to survive. Thus sensitive testingmethods are required for detecting the tumours in early stages or evenin preliminary stages of the cancer such as pre-cancerous stages or theprecursors of malignant cancerous stages.

The most promising methods for early diagnosis of tumours are thoseinvolving molecular markers characteristic for tumours or characteristicfor precursory stages of tumours.

Cancer being a quite heterogeneous disease, multiple regulators of thecell growth can be involved in the genesis of cancer. These regulatoryelements of the cell cycle can be either positive regulators, namedoncogenes when mutated, so that a transformed state is reached, ornegative regulators, named tumour suppressor genes. The number offactors known to be involved in the regulation of the cell cycle andpotentially being causative agents in the development of cancer exceeds100 up to know and is still increasing.

The molecules being involved in the emergence of the cancerous state ofa cell can be used to discriminate between cancer cells and normalissue. Thus cancerous issue can be detected by detecting moleculescharacteristic for the cancer cells. This turns out to be sophisticateddue to the large number of molecules potentially being involved incausing cancer.

For improved diagnosis of tumours there is a need for new markermolecules for use in diagnosis of carcinomas and their precursorlesions, that enable for specific early detection and give theopportunity to treat the disorders at an early stage.

The present invention provides DNase nucleic acids and polypeptides forthe use in detection of carcinomas and their precursor lesions.According to the present invention these molecules may be used asmolecular markers that allow for comprehensive detection of carcinomasand their precursor lesions as e.g. gastrointestinal tract lesions,respiratory tract lesions, etc. even at early stages. Furthermore amethod for the detection of carcinomas and their precursor lesions isprovided.

During the experiments leading to the present Invention it could beshown, that DNase molecules may serve as molecular markers for thedetection of carcinomas and their precursor lesions. Diagnostic value ofDNase nucleic acid or polypeptides for detection of carcinomas and theirprecursor lesions is not published up to date.

Disclosure concerning the mutation of DNase in tumours may be found. Yetthere is no hint as to the use of DNase molecules for the detection anddiagnosis of carcinomas and their precursor lesions.

Investigation on the expression of DNase in carcinomas and theirprecursor lesions and in different stages of tumours elucidated its usefor diagnostic and prognostic purposes. Thus the present invention isbased on the inventors findings shown in the examples given below, thatthe level of expression of DNase nucleic acids as well as of thepolypeptides encoded by these DNase nucleic acids in samples allows todiagnose and grade carcinomas and their precursor lesions such as e.g.gastrointestinal lesions, lesions of the respiratory tract, to predictthe course of the disease and to follow up the disease after initialtherapy.

The inventors could show, that in immunochemical procedures DNase may bedetectable especially in specific subcellular regions such as e.g. inthe nucleus. In the case of DNase X it could be shown, that differentialstaining patterns in immuno-histochemical procedures may depend on therespective binding agents employed in the experiments. It could beshown, that DNase X may be detected at equal levels in Western blot orELISA assays from tumour and normal tissues. In contrast the sametissues render specific nuclear staining patterns for DNase X in tumoursamples and lack staining in normal control samples. Furthermore theinventors found that detection of DNAse activity in body fluids may beused for the identification of individuals having cancers or cancerprecursors.

This may be due to masking of the epitope, that is recognized by theemployed antibody in normal tissue. In tumour tissue the epitope isunmasked especially in the cellular nucleus.

The present invention provides methods for the detection of carcinomasand their precursor lesions comprising the detection of one or moreDNase molecules in a biological sample. The detection of DNase moleculesin the course of the method according to the present invention maycomprise the detection of the level of DNase molecules in biologicalsamples, the detection of the presence or absence of DNase molecules inbiological samples or the determination of the localization of DNasemolecules e.g. in cells.

In one aspect the method according to the present invention isespecially useful for early detection of disorders associated withabnormal cell proliferation such as e.g. colorectal lesions and fordetection of disseminated tumour cells In the course of diagnosis ofminimal residual disease. The method for detection of said carcinomasand their precursor lesions may comprise the detection of the (subcellular) localization of DNase molecules, the detection of the presenceor absence and/or the level of DNase molecules or the detection theaccessibility (detectability) of specific epitopes of DNase molecules inbiological samples. This method may e.g. employ minimally invasive ornon-invasive procedures for obtaining the sample.

In another aspect of the present invention the above mentioned detectionmethods of DNase polypeptides and/or DNase nucleic acids may be used asmolecular markers in the course of staging, assessment of prognosis,monitoring and the design of a strategy of tumour therapeutics.

The present invention furthermore provides DNase nucleic acids andpolypeptides for use in the detection of carcinomas and their precursorlesions, such as e.g. colorectal lesions, lung cancer, gastric cancer,oesophageal cancer, breast cancer, cervical cancer etc.

The present invention also provides kits, such as diagnostic kits orresearch kits, for the detection of the DNase polynucleotides or DNasepolypeptides or comprising DNase polynucleotides, DNase polypeptides orbinding agents specifically binding to DNase polypeptides orpolynucleotides for use in the detection of carcinomas and theirprecursor lesions.

One aspect of the present invention is a method for therapy of disordersassociated with abnormal cell proliferation. In this aspect theinventive DNase polypeptides and/or polynucleotides may be administeredto individuals suffering from said disorders in the course ofimmuno-therapy or gene-therapy. One or more DNase nucleic acids and/orpolypeptides may be used for therapy of carcinomas and their precursorlesions alone or in combination with other molecules.

Yet another aspect of the present invention are pharmaceuticalcompositions containing DNase polypeptides and/or DNase polynucleotidesdisclosed herein alone or combination with one or more other therapeuticor diagnostic agents and/or carrier or adjuvant substances.

It is yet another aspect of the present invention to provide methods foridentification of molecules binding to the nucleic acids andpolypeptides of the present invention as well as of activators andinhibitors of the expression of the genes of the present invention. Alsoa method for the identification of drug candidates for the therapy ofcarcinomas and their precursor lesions is provided.

DNase molecules for use In the context of the present invention compriseDNase I (AJ298844), DNase II (AB004574), DNase I-like 1 (DNase X)(NM_(—)006730), DNase I-like 2 (AK098028), DNase I-like 3 (also calledDNase gamma) (AF047354), caspase activated DNase (AB013918), DNaseKIAA0218 (D86972), DNase II-like DNase (AF274571), DFF-45 (AF087573) andother known DNases.

DNase molecules as used in the context of the present invention maycomprise nucleic acids, polynucleotides, proteins, polypeptides orpeptides. On the level of nucleic acids the marker molecules may be DNAor RNA comprising genomic DNA, cDNA, and RNA such as mRNA or hnRNA.

Generally accessibility as used herein may comprise the localization ofa particular region (the epitope) of a macromolecule on a surface, suchthat second or third molecules may get in contact or interaction to thatregion. Any method for the determination of the accessibility of aspecific region of macromolecules may be employed in the methodsaccording to the present invention. Such methods may e.g. comprisephysical methods, such as e.g. spectroscopy, crystallography, etc.,chemical methods, such as e.g. derivatizaton of functional groups in themacromolecules, crosslinking between neighbour regions in macromoleculesetc. or application of binding agents e.g. in immunochemical procedures.

In certain embodiments accessibility of an epitope of a molecule may bedetermined by methods employing e.g. binding agents. In certain aspectsof the present invention an epitope is said to be accessible, if bindingagents specifically directed against said epitope may bind to andrecognize the epitope in a sample. Inversely the epitope is said to bemasked or inaccessible, if specific binding agents may not bind to theepitope.

Expression as used according to the present invention may comprise forexample expression of proteins. The transcription to RNA and thus thelevel of mRNA may also be understood to be expression according to thepresent invention.

The expression of a compound is said to be significantly alteredaccording to the present invention, if the level of expression differsby more than 30%. The alteration of the expression may comprise forexample elevated expression or reduced expression of said compound.Another aspect of the altered expression may be an alteration in a way,that the compound is expressed under non wild-type circumstances. Thismay comprise, that the compound is for example expressed in situations,that naturally suppress the expression, or is not expressed insituations, that naturally induce the expression of the compound.

Alteration of the expression as used herein may also comprise analteration in the transcription pattern of a gene. E.g. the alterationof the transcripton pattern may comprise alternative splicing of thegene. The alterations in the transcription pattern may influence thepolypeptides translated from the altered transcripts or may berestricted to untranslated regions. The alteration in the transcriptionpattern of a gene may comprise use of novel exons In the transcripts,deletions of exons in the transcripts or the variation in the ratios ofdifferent splicing variants in cells. Thus alterations intranscriptional patterns of genes as used herein may comprise theproduction of nucleic acids such as e.g. mRNA, cDNA etc. containingadditional stretches of nucleic acid sequences compared to wild typenucleic acids occurring in control tissues.

Alternatively the nucleic acids produced by alternative splicingpatterns may produce nucleic acids missing stretches of nucleic acidsequences present in wild type polynucleotides. The presence ofadditional stretches may occur simultaneously with the absence oforiginal sequence-stretches in single transcripts. Alterations in theexpression of genes as used in the context of the present invention mayalso comprise an alteration in the level of expression of splicingvariants of genes. This may include increased or decreased expression ofparticular splicing variants as well as expression of variants notpresent in wild type issue or the absence of expression of splicingvariants present in wild type tissue. In one embodiment the alterationof the expression of the splicing variants may comprise the alterationof the ratios of different splicing variants in said tissue.

Nucleic acids as used in the context of the present invention arepreferably polynucleotides or fragments thereof. Preferredpolynucleotides comprise at least 20 consecutive nucleotides, preferablyat least 30 consecutive nucleotides and more preferably at least 45consecutive nucleotides, that are identical, share sequence homology orencode for identical, or homologous polypepbdes, compared to thepolypeptides associated with the proliferative disorders disclosedherein. The nucleic acids according to the present invention may also becomplementary to any of said polynucleotides. Polynucleotides may forexample include single-stranded (sense or antisense) or double-strandedmolecules, and may be DNA (genomic, cDNA or synthetic) or RNA. RNAmolecules comprise as well hnRNA (containing introns) as mRNA (notcontaining introns). According to the present invention thepolynucleotides may also be linked to any other molecules, such assupport materials or detection marker molecules, and may, but need not,contain additional coding or non-coding sequences.

The DNase polynucleotides for use in a method according to the presentinvention may be native sequences or variants thereof. The variants maycontain one or more substitutions, additions, deletions and/orinsertions such that the immunogenicity of the encoded polypeptide isnot diminished, relative to the respective native DNase proteins. Thevariants show preferably 65-70%, more preferably at least 80% and mostpreferably at least 90% of sequence identity to the native nucleic acidmolecules used in the methods according to the present invention. In oneembodiment of the invention the variants show sequence identity of atleast 65% to 99% or any value in between to the native DNase nucleicacids. In another embodiment of the invention the variants show sequencehomologies of about 60, 65, 70, 75, 80, 85, 90, 95 or even 100%. Methodsfor determination of sequence similarity are known to those of skill inthe art.

In one embodiment of the present invention a variant of DNase moleculesmay be employed, that is altered in a way, that interaction to naturalligands or binding partners is impaired.

One example for detecting the similarity of sequences can be carried outusing the FastA and/or BlastN bioinformatics software accessible on theHUSAR server of the DKFZ Heidelberg.

Furthermore DNase nucleic acids for use in the methods according to thepresent invention are all polynucleotides, which hybridise to probesspecific for the sequences disclosed herein under stringent conditions.Stringent conditions applied for the hybridisation reaction are known tothose of ordinary skill in the art and may be applied as described inSambrook et al. Molecular cloning: A Laboratory Manual, 2^(nd) Edition,1989.

The present invention also employs polynucleotides, that due to thedegeneracy of the genetic code encode the DNase polypeptides nativelyencoded by the disclosed DNase nucleic acids while not showing thepercentage of sequence homology as described above within the nucleicacid sequence. Such nucleic acids might arise by changing the codonspresent in the disclosed sequences by degenerate codons and so preparinga synthetic nucleic acid. In certain special embodiments the codons maybe adjusted to the common codon usage of an appropriate transgenic hostorganism such as e.g. yeast, mice, rats, etc.

The DNase nucleotide sequences used according to the present inventionmay be joined to a variety of other nucleic acid sequences using theknown recombinant DNA techniques. The sequences may for example becloned into any of a variety of cloning vectors, such as plasmids,phagemids, lambda phage derivatives and cosmids. Furthermore vectorssuch as expression vectors, replication vectors, probe generationvectors and sequencing vectors may be joined with the sequencesdisclosed herein. Sequences of special interest, that could be cloned tothe nucleic adds according to the present invention are for example noncoding sequences and regulatory sequences including promoters, enhancersand terminators.

In certain embodiments of the present invention one or more of thenucleic acid sequences encoding DNase polypeptides may be joined. Thismay be especially useful for therapeutic purposes or for the expressionof recombinant proteins. In these embodiments 2, 3, 4, 5, 6, 7, 8, 9, 10or even more different or even identical DNase nucleic acids may bejoined together in one nucleic acid molecule.

In a preferred embodiment DNase polynucleotides may be formulated such,that they are able to enter mammalian cells and to be expressed in saidcells. Such formulations are especially useful for therapeutic purposes.The expression of nucleic add sequences in target cells may be achievedby any method known to those skilled in the art The nucleic acids mayfor example be joined to elements that are apt to enable theirexpression in a host cell. Such elements may comprise promoters orenhancers, such as CMV-, SV40-, RSV-, metallothionein I- orpolyhedrin-promoters respectively CMV- or SV40-enhancers. Possiblemethods for the expression are for example incorporation of thepolynucleotides into a viral vector including adenovirus,adeno-associated virus, retrovirus, vaccinia virus or pox virus. Viralvectors for the purpose of expression of nucleic acids in mammalian hostcells may comprise pcDNA3, pMSX, pKCR, pEFBOS, cDM8, pCEV4 etc. Thesetechniques are known to those skilled in the art.

Other formulations for administration in therapeutic purposes includecolloidal dispersion systems such as for example macromoleculecomplexes, microspheres, beads, micelles and liposomes.

Generally, by means of conventional molecular biological processes it ispossible (see, e.g., Sambrook et al., supra) to introduce differentmutations into the nucleic acid molecules of the invention. As a resultthe inventive tumour associated DNase polypeptides or polypeptidesrelated thereto with possibly modified biological properties aresynthesized. One possibility is the production of deletion mutants inwhich nucleic acid molecules are produced by continuous deletions fromthe 5′- or 3′-terminal of the coding DNA sequence and that lead to thesynthesis of DNase polypeptides that are shortened accordingly. Anotherpossibility is the introduction of single-point mutation at positionswhere a modification of the amino aid sequence influences, e.g., theproliferation specific properties. By this method muteins can beproduced, for example, that possess a modified Km-value or that are nolonger subject to the regulation mechanisms that normally exist in thecell, e.g. with regard to allosteric regulation or covalentmodification, or altered binding-, dimerization-, inter- orintramolecule interaction properties. Such muteins might also bevaluable as therapeutically useful agonists or antagonists of the DNasemolecules used in the methods according to the present invention.

For the manipulation in prokaryotic cells by means of geneticengineering the DNase nucleic acid molecules of the invention or partsof these molecules can be introduced Into plasmids allowing amutagenesis or a modification of a sequence by recombination of DNAsequences. By means of conventional methods (cf. Sambrook et al., supra)bases can be exchanged and natural or synthetic sequences can be added.In order to link the DNA fragments with each other adapters or linkerscan be added to the fragments. Furthermore, manipulations can beperformed that provide suitable cleavage sites or that removesuperfluous DNA or cleavage sites if insertions, deletions orsubstitutions are possible, in vitro mutagenesis, primer repair,restriction or ligation can be performed. As analysis method usuallysequence analysis, restriction analysis and other biochemical ormolecular biological methods are used.

The DNase polypeptides encoded by the various variants of the DNasenucleic acid molecules of the invention show certain commoncharacteristics, such as molecular weight, immunological reactivity orconformation or physical properties like the electrophoretic mobility,chromatographic behaviour, sedimentation coefficients, solubility,spectroscopic properties, stability, pH optimum, temperature optimum.

The invention furthermore employs vectors containing the inventivetumour associated DNase nucleic acid molecules. Preferably, they areplasmids, cosmids, viruses, bacteriophages and other vectors usuallyused in the field of genetic engineering. Vectors suitable for use inthe present invention include, but are not limited to the T7-based dualexpression vectors (expression in prokaryotes and in eucaryotes) forexpression in mammalian cells and baculovirus-derived vectors forexpression in insect cells. Preferably, the DNase nucleic acid moleculefor use in the method according to the invention is operatively linkedto the regulatory elements in the recombinant vector of the inventionthat guarantee the transcription and synthesis of an mRNA in prokaryoticand/or eucaryotic cells that can be translated. The nucleotide sequenceto be transcribed can be operatively linked to a promoter like a T7,metallothionein I or polyhedrin promoter.

In a further embodiment, the present invention makes use of recombinanthost cells transiently or stably containing DNase nucleic acidmolecules. A host cell is understood to be an organism that is capableto take up in vitro recombinant DNA and, if the case may be, tosynthesize the polypeptides encoded by the nucleic acid molecules of theinvention. Preferably, these cells are prokaryotic or eucaryotic cells,for example mammalian cells, bacterial cells, plant cells, insect cellsor yeast cells. The host cells for use in the invention are preferablycharacterized by the fact that the Introduced DNase nucleic acidmolecule either is heterologous with regard to the transformed cell,i.e. that it does not naturally occur in these cells, or is localized ata place in the genome different from that of the corresponding naturallyoccurring DNase sequence.

A further embodiment of the invention relates to the use of apolypeptide exhibiting a biological property of DNases and being encodedby the known DNase nucleic acid molecules.

These proteins or polypeptides may be produced by any suitable methodincluding methods, whereby, e.g., a host cell is cultivated underconditions allowing the synthesis of the DNase polypeptide and the DNasepolypeptide is subsequently isolated from the cultivated cells and/orthe culture medium.

Isolation and purification of the recombinantly produced polypeptide maybe carried out by conventional means including preparativechromatography and affinity and immunological separations using, e.g.,an antibody directed against the inventive tumour associated markerproteins, or, e.g., can be substantially purified by the one-step methoddescribed in Smith and Johnson, Gene 67; 31-40 (1988).

The polypeptides for use In the present invention however, not onlycomprise recombinantly produced DNase polypeptides but include isolatednaturally occurring DNase polypeptides, synthetically produced DNasepolypeptides, or polypeptides produced by a combination of thesemethods. Means for preparing such polypeptides or related polypeptidesare well understood in the art These polypeptides are preferably in asubstantially purified form.

The production of a DNase polypeptide for use in a method according tothe present invention may for example be carried out in a cell free invitro transcription and/or translation system. Such systems are known tothose of ordinary skill in the art. One example may comprise an introtranslation system as provided by Roche molecular Biochemicals' Rapidtranslation System.

DNase (poly)peptides as used in methods according to the presentinvention may comprise amino acid chains of any length, including fulllength proteins, wherein the amino acid residues are linked by covalentpeptide bonds.

DNase peptides for use in the detection or treatment of carcinomas andtheir precursor lesions as disclosed in the context of the presentinvention shall comprise polypeptides of lengths of at least 4 aminoacids. These DNase peptides may for example comprise 4 to 50 amino acidsor any number of amino acids in between. In another embodiment of thepresent invention the peptides may comprise polypeptides with more than50 amino acids. These DNase polypeptides for use in the methods of thepresent invention may for example comprise 50, 100, 500, 750, 1000 aminoacids or any number of amino adds in between and may comprise proteins,or fragments thereof, and/or fusion- or chimeric proteins comprising onor more additional heterologous sequences. The additional sequences maybe derived from the native DNase proteins or may be heterologous, andsuch sequences may (but need not) be immuno-reactive and/or antigenic.As detailed below, such polypeptides may be isolated from tumour issueor prepared by synthetic or recombinant means.

As used herein, a polypeptide exhibiting biological properties of DNasepeptides for use in the methods disclosed herein is understood to be apolypeptide having at least substantially the same immunogenicproperties, i.e. is still capable of binding an antibody directedagainst a DNase polypeptide, e.g. comprises at least one immunogenicepitope of a DNase polypeptide.

Peptides for use in a method as disclosed herein may be e.g. immunogenicpolypeptides. This requires, that the polypeptides may stimulate Immuneresponses in host organisms either in the form the polypeptides adopt intheir natural environment and/or especially in the form the polypeptidesadopt after processing by the cellular antigen processing and presentingmachinery.

Immunogenic portion as used above is a portion of a protein, that isrecognized by a B-cell and/or T-cell surface antigen receptor. Theimmunogenic portions comprise at least 4 amino acid residues, at least10 amino acid residues or at least 15 amino acid residues of the proteindisclosed herein. In one embodiment of the present invention, particulardomains of the protein, such as for example transmembrane domains orN-terminal leader sequences have been deleted.

The immunogenic portions according to the present invention react withantisera or specific antibodies in the same or nearly same intensity asthe native full length proteins. The immunogenic portions are generallyidentified using the techniques well known in the art Possibletechniques are for example screening of the polypeptides for the abilityto react with antigen-specific antibodies, antisera and/or T-cell linesor clones.

Suited immunogenic portions for DNase X may e.g. comprise the peptides:

-   -   71-90: RELNRFDGSGPYSTLSSPQL    -   207-224: HWVIADGEDTTVRASTHC    -   187-206: CASLTKKRLDKLELRTEPGF    -   225-241: TYDRWLHGERCRSLLH    -   254-269: LTEEEALNISDHYPVE    -   110-126: VLSSYVYNDEDDVFARE

These immunogenic sequences for DNase X shall be examples forimmunogenic regions and shall not be construed to limit the scope of thepresent invention. For all DNases the immunogenic regions for use in amethod according to the present invention may be determined by anysuitable method. The methods for determining the respective immunogenicregions in the particular DNase molecules are known to those of skill inthe art.

In certain embodiments of the present invention DNase polypeptides maycomprise fusion or chimeric polypeptides containing sequences disclosedherein. Fusion proteins comprise the polypeptide according to thepresent invention together with any second and further polypeptides,such as e.g. one or more polypeptides of the same sequence or of anothersequence. Heterologous polypeptides may comprise e.g. enzymes, receptormolecules, antigens, antigenic or immunogenic epitopes or fragments,antibodies or fragments thereof, signalling polypeptides or signaltransducing polypeptides, labelled polypeptides etc. The immunogenicprotein may for example be capable of eliciting a recall response.Examples of such proteins include tetanus, tuberculosis and hepatitisproteins (see, for example, Stoute et al. New Engl. J. Med., 336:86-91(1997)). For use in pharmaceutical compositions fusion proteinscomprising serum albumin or fragments thereof may be useful in certainembodiments of the present invention.

In one embodiment of the invention the fusion peptides may beconstructed for enhanced detection or purification of the polypeptides,or of complexes of the DNase polypeptides with the respectiveimmunological entities according to the present invention. For thepurpose of purification tags, such as e.g. his-tags, myc-tags etc. maybe added to the polypeptides. For the purpose of detection antigenicportions, enzymes, chromogenic sequences etc. may be fused to thepolypeptides. The fusion proteins of the present invention may (but neednot) include a linker peptide between the first and second polypeptides.

A peptide linker sequence may be employed to separate the first and thesecond polypeptides by a distance sufficient to ensure, that eachpolypeptide folds into its secondary and tertiary structures. Such apeptide linker sequence is incorporated into the fusion protein usingstandard techniques well known in the art. Suitable peptide linkersequences may be chosen based on the following factors: (1) theirability to adopt a flexible extended conformation; (2) their inabilityto adopt a secondary structure that could interact with functionalepitopes on the first and second polypeptides; and (3) the lack ofhydrophobic or charged residues that might react with the polypeptidefunctional epitopes.

Preferred peptide linker sequences contain Gly, Asn and Ser residues.Other near neutral amino acids, such as Thr and Ala may also be used inthe linker sequence. Amino acid sequences which may be usefully employedas linkers include those disclosed in Maratea et al., Gene 40:3946,1985; Murphy et al., Proc. Natl. Acad. Sci. USA 83:8258-8262, 1986; U.S.Pat. No. 4,935,233 and U.S. Pat. No. 4,751,180. The linker sequence maybe from 1 to about 50 amino acids in length. Peptide sequences are notrequired when the first and second polypeptides have non-essentialN-terminal amino acid regions that can be used to separate thefunctional domains and prevent steric interference.

The DNase polypeptides for use in a method according to the presentinvention comprise also variants of the native DNase proteins. Thesevariants may differ from the native protein in one or more alterationssuch as substitutions, deletions, additions and/or insertions. Theimmuno-reactivity of the variants according to the present invention isnot substantially diminished compared to the native DNase proteins. In apreferred embodiment of the invention the immuno-reactivity isdiminished less than 50% in a more preferred embodiment theimmuno-reactivity Is diminished less than 20% compared to the nativepolypeptides. In one embodiment the immuno-reactivity is diminished lessthan 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or any value inbetween. In certain embodiments the immuno-reactivity of the variants isreduced by even more than 50%.

In one embodiment DNase variants may be deficient in one or moreportions, such as for example N-terminal leader sequences, transmembranedomains or small N- and/or C-terminal sequences. The variants exhibit60%, 65% or 70%, more preferably at least 75%, 80%, 85% or 90% and mostpreferably at least 92.5%, 95%, 97.5%, 98%, 98.5%, 99% or 99.5% identityto the DNase polypeptides disclosed according to the present invention.

The variants of the present invention are preferably conservativesubstitutions, so that the amino acids changed are substituted for aminoacids with similar properties. The properties concerned may includepolarity, charge, solubility, hydrophobicity, hydnophilicity and/oramphipathic nature of the amino acid residues. The variants disclosedherein may also comprise additional terminal leader sequences, linkersor sequences, which enable synthesis, purification or stability of thepolypeptdes in an easier or more comfortable way.

The DNase (poly)peptides for use in a method according to the presentinvention may be produced by any method known to those of skill in theart. E.g. the polypeptdes may be isolated from cells or organismsexpressing the polypeptides, may be produced recombinantiy inrecombinant host cells or may be synthesized chemically by the methodscommonly applied for synthesis of polypeptdes.

The term binding agent as used herein comprises a variety of substancessuch as oligopeptides, antibodies, peptdiomimetc molecules comprisingantigen binding oligopeptides, nucleic acids, carbohydrates, organiccompounds, etc. Antibody according to the present invention preferablyrelates to antibodies which consist essentially of pooled monodonalantibodies with different epitopic specificites, as well as distinctmonoclonal antibody preparations. Monoclonal antibodies are made from anantigen containing fragments of the polypeptides of the invention bymethods well known to those skilled in the art (see, e.g., Kohler etal., Nature 256 (1975), 495). As used herein, the term “antibody” (Ab)or “monoclonal antibody” (Mab) is meant to include intact molecules aswell as antibody fragments (such as, for example, Fab and F(ab′) 2fragments) which are capable of specifically binding to protein. Fab andf(ab′)2 fragments lack the Fc fragment of intact antibody, clear morerapidly from the circulation, and may have less non-specific tissuebinding than an intact antibody. (Wahl et al., J. Nucl. Med. 24: 316-325(1983)). Thus, these fragments are preferred, as well as the products ofa Fab or other immuno-globulin expression library. Moreover, antibodiesof the present invention include chimerical, single chain, and humanizedantibodies.

Binding agents used according to the present invention may for examplebe employed for the inhibition of the activity of the inventive DNasepolypeptides. In this respect the term “binding agents” relates toagents specifically binding to the DNase polypeptides transcribed fromthe novel tumour associated nucleic acids and thus inhibiting theactivity of said polypeptide. Such binding agents may for examplecomprise nucleic acids (DNA, RNA, PNA etc.), polypeptides (antibodies,receptors, antigenic fragments, oligopeptides), carbohydrates, lipids,organic or inorganic compounds (metal-ions, sulphur compounds, boranes,silicates, reducing agents, oxidizing agents). The binding agents maypreferably interact with the polypeptide by binding to epitopes, thatare essential for the biological activity. The interaction may bereversible or Irreversibly. The binding may be non-covalent or evencovalent binding to the polypeptide. Furthermore the binding agents mayintroduce alterations to the DNase polypeptide, that alter or diminishthe biological activity of the inventive DNase polypeptide.

For certain purposes, e.g. diagnostic methods, the antibody or bindingagent of the present invention may be detectably labelled, for example,with a radioisotope, a bioluminescent compound, a chemiluminescentcompound, a fluorescent compound, a metal chelate, a biologicallyrelevant binding structure such as biotin or digoxygenin or an enzyme.Furthermore any method suitable for the detection of the intermolecularinteraction may be employed.

The antibody or antigen-binding agent is said to react specifically, ifit reacts at a detectable level with a DNase protein as used in a methodaccording to the present invention herein, and does not significantlyreact with other proteins. The antibodies according to the presentinvention may be monoclonal or polyclonal antibodies. Other moleculescapable of binding specifically may be for example antigen-bindingfragments of antibodies such as Fab fragments, RNA molecules orpolypeptides. According to the present invention binding agents may beused isolated or in combination. By means of combination it is possibleto achieve a higher degree of sensitivity.

In certain embodiments binding agents may exhibit selectivespecificities for the various DNase polypeptides that may be used in themethods according to the present invention. These binding agents maye.g. be defined by epitopic specificity. The specificity may e.g. bechosen in a way to ensure that only one polypeptide product of the DNasegene is recognized by the respective binding agent.

The antibodies or binding agents useful for the methods according to thepresent invention may comprise further binding sites for eithertherapeutic agents or other polypeptides or may be coupled to saidtherapeutic agents or polypeptides. Therapeutic agents may comprisedrugs, toxins, radio-nuclides and derivatives thereof. The agents may becoupled to the binding agents either directly or indirectly for exampleby a linker or carrier group. The linker group may for example functionin order to enable the coupling reaction between binding agent andtherapeutic or other agent or the linker may act as a spacer between thedistinct parts of the fusion molecule. The linker may also be cleavableunder certain circumstances, so as to release the bound agent under saidconditions. The therapeutic agents may be covalently coupled to carriergroups directly or via a linker group. The agent may also benon-covalently coupled to the carrier. Carriers that can be usedaccording to the present invention are for example albumins,polypeptides, polysaccharides or liposomes.

The antibody used according to the present invention may be coupled toone or more agents. The multiple agents coupled to one antibody may beall of the same species or may be several different agents bound to oneantibody.

The invention makes use of transgenic non-human animal such astransgenic mice, rats, hamsters, dogs, monkeys, rabbits, pigs, C.elegans and fish such as torpedo fish comprising a DNase nucleic acidmolecule or vector of the invention, preferably wherein said DNasenucleic acid molecule or vector may be stably integrated into the genomeof said non-human animal, preferably such that the presence of saidDNase nucleic acid molecule or vector leads to the expression of theDNase polypeptide (or related polypeptide), or may otherwise betransiently expressed within the non-human animal. Said animal may haveone or several copies of the same or different nucleic acid moleculesencoding one or several forms of DNase polypeptide or mutant formsthereof. This animal has numerous utilities, including as a researchmodel for the regulation of cell proliferation and differentiation andtherefore, presents a novel and valuable animal in the development oftherapies, treatment, etc. for diseases caused by deficiency or failureof the DNase protein involved in the development of cell proliferativedisorders, e.g., tumours. Accordingly, in this instance, the non-humanmammal is preferably a laboratory animal such as a mouse or rat.

In certain embodiments, the transgenic non-human animal furthercomprises at least one inactivated wild type allele of the correspondinggene encoding the inventive DNase polypeptide. This embodiment allowsfor example the study of the interaction of various mutant forms ofDNase polypeptides. All the applications that have been herein beforediscussed with regard to a transgenic animal also apply to animalscarrying two, three or more transgenes.

In the methods according to the present invention it might be alsodesirable to inactivate protein expression or function at a certainstage of development and/or life of the transgenic animal. This can beachieved by using, for example, tissue specific, developmental and/orcell regulated and/or inducible promoters which drive the expression of,e.g., an antisense or ribozyme directed against the RNA transcriptencoding the inventive DNase encoding mRNA; see also supra. A suitableinducible system is for example tetracycline-regulated gene expressionas described, e.g., by Gossen and Bujard (Proc. Natl. Acad. Sci. 89 USA(1992), 5547-5551) and Gossen et al. (Trends Biotech. 12 (1994), 58-62).Similar, the expression of the mutant inventive tumour associatedprotein may be controlled by such regulatory elements.

Furthermore, the invention in certain embodiments makes use of atransgenic mammalian cell which contains (preferably stably integratedinto its genome or transiently introduced) a DNase nucleic acid moleculeor part thereof, wherein the transcription and/or expression of thenucleic acid molecule or part thereof leads to reduction of thesynthesis of a native DNase molecule. In a preferred embodiment, thereduction is achieved by an antisense, sense, ribozyme, co-suppressionand/or dominant mutant effect. “Antisense” and “antisense nucleotides”means DNA or RNA constructs which block the expression of the naturallyoccurring gene product. In another embodiment the native nucleic acidsequence coding for the DNase polypeptide may be altered or substitutedby a variant of said nucleic acid sequence, e.g. by means ofrecombination, thus rendering the DNase gene non functional. Thus anorganism lacking the DNase activity may be produced according to knockout experiments.

In certain embodiments transgenic non-human animals with a reduced levelof DNase protein may be useful. Techniques how to achieve this are wellknown to the person skilled in the art. These include, for example, theexpression of antisense-RNA, ribozymes, of molecules which combineantisense and ribozyme functions and/or of molecules which provide for aco-suppression effect When using the antisense approach for reduction ofthe amount of the inventive tumour associated marker proteins in cells,the nucleic acid molecule encoding the antisense-RNA is preferably ofhomologous origin with respect to the animal species used fortransformation. However, it is also possible to use nucleic acidmolecules which display a high degree of homology to endogenouslyoccurring nucleic acid molecules encoding a DNase protein. In this casethe homology is preferably higher than 75%, 80% or 85%, particularlyhigher than 90%, 91%, 92%, 93% or 94% and still more preferably higherthan 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5%. Thereduction of the synthesis of a DNase polypeptde for use in a methodaccording to the invention in the transgenic mammalian cells can resultin an alteration in, e.g., degradation of endogenous proteins. Intransgenic animals comprising such cells this can lead to variousphysiological, developmental and/or morphological changes.

Thus, the present invention also makes use of transgenic non-humananimals comprising the above-described transgenic cells. These may show,for example, a deficiency in regulation of cell proliferation and/ordifferentabon compared to wild type animals due to the stable ortransient presence of a foreign DNA resulting in at least one of thefollowing features:

-   -   (a) disruption of (an) endogenous gene(s) encoding a DNase;    -   (b) expression of at least one antisense RNA and/or ribozyme        against a transcript comprising a DNase nucleic acid;    -   (c) expression of a sense and/or non-translatable mRNA of a        DNase nucleic acid;    -   (d) expression of an antibody directed against a DNase        polypeptde;    -   (e) incorporation of a functional or non-functional copy of the        regulatory sequence of a DNase; or    -   (f) incorporation of a recombinant DNase molecule or vector        containing a DNase nucleic acid.

Methods for the production of a transgenic non-human animal for use inthe present invention, preferably a transgenic mouse, are well known tothe person skilled in the art. Such methods, e.g., comprise theintroduction of a nucleic acid molecule or vector into a germ cell, anembryonic cell, stem cell or an egg or a cell derived thereof. Thenon-human animal can be used in accordance with a screening methoddescribed herein and may be a non-transgenic healthy animal, or may havea disorder, preferably a disorder caused by at least one mutation in aDNase protein and/or gene.

Such transgenic animals are well suited for, e.g., pharmacologicalstudies of drugs in connection with mutant forms of the above describedinventive tumour associated marker polypeptide. Production of transgenicembryos and screening of those can be performed, e.g., as described byA. L. Joyner Ed., Gene Targeting, A Practical Approach (1993), OxfordUniversity Press. The DNA of the embryonal membranes of embryos can beanalysed using, e.g., Southern blots with an appropriate probe,amplification techniques based on nucleic acids (e.g. PCR) etc.; seesupra.

Another aspect of the present Invention is a pharmaceutical compositionfor use in the treatment of carcinomas and their precursor lesions. TheDNase polypeptides, DNase polynucleotides and DNase binding agents (esp.antibodies) used according to the present invention may be Incorporatedinto pharmaceutical or immunogenic compositions.

The pharmaceutical compositions may be administered by any suitable wayknown to those of skill in the art. The administration may for examplecomprise injection, such as e.g., intracutaneous, intramuscular,intravenous or subcutaneous injection, intranasal administration forexample by aspiration or oral administration. A suitable dosage toensure the pharmaceutical benefit of the treatment should be chosenaccording the parameters, such as age, sex, body weight etc. of thepatent, known to those of skill in the art.

The pharmaceutical compositions comprise said compounds and aphysiologically acceptable carrier. The type of carrier to be employedin the pharmaceutical compositions of this invention, will varydepending on the mode of administration. For parenteral administration,such as subcutaneous injection, the carrier preferably comprises water,saline, alcohol, a lipid, a wax and/or a buffer. For oraladministration, any of the above carriers or a solid carrier, such asmannitol, lactose, starch, magnesium stearate, sodium saccharine,talcum, cellulose, glucose, sucrose, and/or magnesium carbonate, may beemployed. Biodegradable microspheres (e.g., polylactic glycolide) mayalso be employed as carriers for the pharmaceutical compositions of thisinvention. Suitable biodegradable microspheres are disclosed, forexample, in U.S. Pat. Nos. 4,897,268 and 5,075,109.

A pharmaceutical composition for use in a method according to thepresent invention may for example contain DNA, that codes for one ormore DNase polypeptides. The DNA may be administered in a way thatallows the polypeptides to be generated in situ. Suitable expressionsystems are known to those skilled in the art. In another embodiment ofthe invention the DNase nucleic acids may be for example ant-senseconstructs. Pharmaceutical compositions may also comprise DNase nucleicacid molecules expressible in a mammalian or human host systemcomprising a viral or other expression system for example an adenoviralvector system.

The DNase nucleic acid may also be administered as a naked nucleic acid.In this case appropriate physical delivery systems, which enhance theuptake of nucleic acid may be employed, such as coating the nucleic acidonto biodegradable beads, which are efficiently transported into thecells. Administration of naked nucleic acids may for example be usefulfor the purpose of transient expression within a host or host cell.

Alternatively the pharmaceutical compositions may comprise one or morepolypeptides. The polypeptides incorporated into pharmaceuticalcompositions may be a DNase polypeptide. Optionally the DNasepolypeptide may be administered in combination with one or more otherknown polypeptides such as for example enzymes, antibodies, regulatoryfactors, such as cyclins, cyclin-dependent kinases or CKIs, or toxins.

DNase polypeptides used in the present invention or fragments thereof,that comprise an immunogenic portion may be used in pharmaceuticalcompositions, wherein the polypeptide e.g. stimulates a responsedirected specifically against tumour cells in the patient. A patent maybe afflicted with disease, or may be free of detectable disease.Accordingly, the DNase compounds may be used to treat cancer or toinhibit the development of cancer. The compounds may be administeredeither prior to or following a conventional treatment of tumours such assurgical removal of primary tumours, treatment by administration ofradiotherapy, conventional chemotherapeutic methods or any other mode oftreatment of the respective cancer or its precursors.

Immunogenic compositions may comprise one or more polypeptides andnon-specific immune-response enhancers, wherein the non-specific immuneresponse enhancer is capable of eliciting or enhancing an immuneresponse to an exogenous antigen. Any suitable immune-response enhancermay be employed in the vaccines of this invention. For example, anadjuvant may be included. Most adjuvants contain a substance designed toprotect the antigen from rapid catabolism, such as aluminium hydroxideor mineral oil, and a non-specific stimulator of immune response, suchas lipid A, Bordetella pertussis or Mycobacterium tuberculosis. Suchadjuvants are commercially available as, for example, Freund'sIncomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit,Mich.) and Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.).

Pharmaceutical compositions and vaccines may also contain other epitopesof tumour antigens, either incorporated into a fusion protein asdescribed above (i.e., a single polypeptide that contains multipleepitopes) or present within a separate polypeptide.

The present invention further provides kits for use in e.g. research ordiagnostic methods. Such kits may contain two or more components forperforming a scientific or diagnostic assay. Components may becompounds, reagents, containers and/or equipment. One component may bean antibody or fragment thereof that specifically binds to a DNasepolypeptide. Additionally the kit may contain reagents, buffers orothers known in the art as necessary for performing the diagnosticassay. Alternatively the research kit or diagnostic kit may containnucleotide probes or primers for the detection of DNASE DNA or RNA. Sucha kit should contain appropriate additional reagents and buffers knownin the art.

A kit according to present invention comprises:

-   -   a) reagents for the detection of the DNase marker molecules    -   b) the reagents and buffers commonly used for carrying out the        detection reaction, such as buffers, detection-markers, carder        substances and others    -   d) a DNase marker sample for carrying out a positive control        reaction.

The reagent for the detection of the DNase marker Includes any agentcapable of binding to the marker molecule. Such reagents may includeproteins, polypeptides, nucleic acids, glycoproteins, proteoglycans,polysaccharides or lipids.

The sample for carrying out a positive control may comprise for exampleDNase nucleic acids in applicable form, such as solution or salt, DNasepeptides in applicable form, tissue section samples or positive cellsexpressing the DNase molecules.

In a preferred embodiment of the invention the detection of the markermolecules is carried out on the level of polypeptides. In thisembodiment the binding agents may be for example antibodies specific forDNase or fragments thereof.

In another embodiment of the test kit the detection of DNase is carriedout on the nucleic acid level. In this embodiment of the invention thereagents for the detection may be for example nucleic acid probes orprimers complementary to said DNase nucleic acids.

Carcinomas and their precursor lesions according to the presentinvention are disorders characterized by abnormal growth properties ofcells or tissues compared to the growth properties of normal controlcells or tissues. The growth of the cells or tissues may be for exampleabnormally accelerated or may be regulated abnormally. Abnormalregulation as used above may comprise any form of presence or absence ofnon wild-type responses of the cells or tissues to naturally occurringgrowth regulating influences. The abnormalities in growth of the cellsor tissues may be for example neoplastic or hyperplastic. In onepreferred embodiment of the invention the tumours are cancers orpre-cancerous conditions of the respiratory tract.

Disorders characterized by abnormal cell proliferation, as used in thecontext of the present invention, may comprise for example neoplasmssuch as benign and malignant tumours, carcinomas, sarcomas, leukemias,lymhomas or dysplasias. Tumours may comprise tumours of the head and theneck, tumours of the respiratory tract, tumours of the gastrointestinaltract, tumours of the urinary system, tumours of the reproductivesystem, tumours of the endocrine system, tumours of the central andperipheral nervous system, tumours of the skin and its appendages,tumours of the soft tissues and bones, tumours of the lymphopoietic andhematopoietic system, breast cancer, prostate cancer, gastrointestinalcancer, colorectal cancer, anogenital cancer etc.

In certain embodiments the disorders are for example adenomas oradenocarcinomas of the colon, disorders of the respiratory tract such asSquamous Cell Lung Carcinoma, Small Cell Lung Carcinoma, Adenocarcinomaof the Lung, Large Cell Lung Carcinoma, Adeno-Squamous Lung Carcinoma,Carcinoid Tumour of the Lung, Bronchial Gland Tumour or (malignant)Mesothelioma, anogenital cancer such as, cervical cancer, vulval cancer,vaginal cancer, cancer of the rectum, cancer of the anus and cancer ofthe penis.

A sample according to the method of the present invention is any sample,that may contain cells, tissues or body fluid. Furthermore any samplepotentially containing the marker molecules to be detected may be asample according to the present invention. Such samples are e.g. blood,plasma, serum, liquor, bone marrow, swabs, washes, secretions,transsudates, exudates, sputum, stool, urine, semen, cell- andtissue-samples, punctuates or biopsies.

Biopsies as used in the context of the present invention may comprisee.g. resection samples of tumours, issue samples prepared by endoscopicmeans or needle biopsies. Furthermore any sample potentially containingthe marker molecules to be detected may be a sample according to thepresent invention.

In one embodiment of the present invention samples comprise cells of theanogenital tract, of the respiratory tract, the gastrointestinal tract(esp. the colorectal tract) or of the skin and its appendages. Incertain embodiments the cells may be cells of the uterine cervix, thevagina, the vulva, the penis, the anus, the rectum, the bronchic tree,the lung, the peritoneum, the peritoneal space, the naso-pharyngealspace, the oral cavity, the colon ascendens, the colon transversum, thecolon descendens, the colon sigmoidum, the pancreas, the smallintestine, the duodenum, the jejunum, the ileum, the caecum, theoesophagus, the stomach, the bile tree, the liver or the skin.

In certain embodiments of the present invention the sample may be ahistological sample, a biopsy, or a cytological sample such as e.g. asmear, a swab, a wash, a body fluid containing cells (sputum, asecretion, saliva, etc.). In certain embodiments of the presentinvention the samples may comprise cells infected by papilloma virus.The samples may in certain embodiments comprise cervical smears,bronchioalveolar lavages, stool, samples obtained by endoscopic meanssuch as e.g. gastroscopy, colonoscopy, bronchioscopy etc.

Preparation of a sample may comprise e.g. obtaining a sample of atissue, of a body fluid, of cells from a patient According to thepresent invention preparation of the sample may also comprise severalsteps of further preparations of the sample, such as preparation ofdissections, preparation of cell suspensions, spreading or applying thecells to be examined onto microscopic slides, preparation of tissuearrays, isolation of polypeptides or nucleic acids, preparation of solidphase fixed peptides or nucleic acids or preparation of beads, membranesor slides to which the molecules to be determined are coupled covalentlyor non-covalently.

A sample for use in the method of the present invention may be obtainedand prepared by any suitable procedure. The samples e.g. may compriseany samples of the content of the gastrointestinal tract (e.g. thestomach, the oesophagus or the bowel) of the respiratory tract (e.g. ofthe naso-pharyngeal space, the bronchus or the bronchioles), theanogenital tract (e.g. the vagina), the urogenital tract (e.g. thebladder or the urethra) the vascular system etc. The sample may beobtained by active excretion of the material by an individual, or may bemay obtained in the course of a surgical, invasive or minimally invasivemedical procedure. For example a stool sample, as used in the context ofthe present invention may be a sample of material contained in the lumenof the colon, which has been obtained by dysters, by colonoscopy,digitally from the rectum or may be obtained from a stool voided by apatient A stool sample according to the present invention may, but neednot contain cells or cell debris of colorectal origin. In one embodimentof the present invention the polypeptides used for the detection of thecolorectal lesions are secreted proteins that may be detected in stoolsamples independent of the presence of cells or cell debris originatingfrom a colorectal lesion within the sample. In one embodiment of thepresent invention the sample may comprise blood, lymph, lymph nodes,bone marrow etc. In another embodiment of the present invention thesample may comprise breast tissue, breast cells, nipple aspirates,ductal ravages or any sample containing cells or cell debris originatingfrom the breast.

In certain embodiments of the present invention sample may refer to therespective material within the body of an individual such as the urine,the stool, the sputum etc. A sample in this context may be e.g. thecontent of the intestine of an individual in vivo. In this embodimentthe (stool, urine, sputum, exudates, semen, secretion) sample need notto be separated from the patient to be subjected to the methodsdisclosed herein.

The method for preparation of the sample may comprise any methodsuitable for assuring accurate detection of the presence or absence oflesions associated with abnormal growth properties. In certainembodiments of the present invention the preparation of the samples maycomprise e.g. picking any portion of a total samples (such as e.g.voided stool, excreted urine, obtained smear, wash, sputum) with anysuitable means such as a spatula, a brush, a spoon, a tip, a cloth, amembrane, a capillary, a syringe, a needle or pin or the like. Forexample the sample preparation may comprise blotting of a portion of thesample (such as e.g. the surface of a stool) to a membrane, a foil, aplastic film or a cloth, picking a portion of the stool by means of aneedle, syringe, capillary, spatula, spoon or the like. Samplepreparation may in certain embodiments comprise swabs from the surfaceof solid or viscous samples (such as e.g. voided stool, certainsecretions, etc) obtained by suitable means like a spatula, a brush, atampon, a cloth, a porous or textile (cotton, cellulose, derivatizedcellulose etc.) device or tip or any other suitable means.

In certain embodiments of the invention any random fraction of a samplemay be suitable for performing the detection method disclosed herein. Incertain other embodiments of the invention a sample may be prepared in away to assure the presence of a representative portion of the totalsample. Such representative portions may be e.g. obtained by the methodsdescribed in U.S. Pat. No. 6,303,304, which shall be incorporated hereinby reference, for stool samples.

In certain special embodiments of the present invention the sample maybe prepared as a monolayer or thin layer preparation of a cytologicalspecimen. The respective methods for preparation of monolayer orthin-layer preparation in cytology are known to those of skill in theart. In one embodiment the preparation may e.g. comprise the ThinPrep™technology. Other methods comprise conventional smears, or methodemploying suspensions of cells for preparation of the cytologicalspecimens.

In certain embodiments of the present invention the a procedure forenriching or purifying the polypeptides and/or polynucleotides ofinterest may be employed. Where the case may be, the identification ofnucleic acids, proteins or peptides in complex samples, which compriseseveral nucleic acids, proteins or peptides, may be enhanced by aseparation procedure of particular molecule species present in thesample. These purification processes may involve purification in themeaning of separating all nucleic acids, protein or peptide componentsof the sample from other components such as lipids, nucleic acids etc.In certain embodiments of the Invention the purification may alsoinvolve the separation of nucleic acids and/or proteins or peptides ofparticular properties from other protein or peptide components withinthe mixture.

Generally the methods for purification of nucleic acids and polypeptidesmentioned herein may be applied in the course of any detection proceduresuitable for the detection of the DNase molecules of the presentinvention. Thus, as the case may be, any detection procedure for thedetection of the marker molecules disclosed herein alone or combinationwith other marker molecules may comprise purification methods fornucleic acids and/or polypeptides as mentioned below. The purificationprocess may be performed at any stage In the course of the totalprocedure e.g. before a detection or amplification reaction,subsequently to a detection or amplification reaction, in a single stepreaction simultaneously to a detection or amplification reaction etc.

Separation of proteins and/or nucleic acids may be carried out by use oftheir physical, chemical or biological properties. Physical parametersused for separation may comprise charge, hydrophobicity, mass, volume,shape or any other physical parameter suitable for separating differentprotein or peptide species. Chemical parameters applicable in separationof proteins comprise the use of reactive groups such as hydroxyl,sulfhydryl or any other reactive or non reactive structure suitable forthe separation of proteins/peptides. Biological parameters which may beused for separating proteins may include enzymatic activity, molecularinteractions such as e.g. binding of biological binding moieties, suchas e.g. ligands or receptors, immunogenicity or any other biologicalproperty suitable for separating different proteins or peptides. Withrespect to nucleic acids biological parameter may especially pertain tohybridisation properties.

All parameters mentioned above may be used independently or in anycombination suitable for separating and purifying nucleic adds, proteinsand/or peptides. In one embodiment a complex sample may be separated byelectrophoretic methods such as agarose gel electrophoresis, PAGE,SDS-PAGE, free flow electrophoresis, capillary electrophoresis,2D-electrophoresis or any other electrophoretic method suitable forseparating nucleic acids, proteins or peptides. In certain embodiments2D-electrophoresis may be used such that the separation in the firstdimension is based on charge (e.g. in a polyacrylamide gel under highvoltage conditions) and the resulting separated proteins or peptides areseparated by their mass (e.g. in a sodium dodecylsulfate polyacrylamidegel in a direction perpendicular to the first dimension). Alternativelythe fist dimension of separation of proteins or peptides may be achievedby isoelectric focussing of the molecules in a pH gradient under highvoltage. In certain embodiments a pulsed field electrophoresis may beapplied for the separation of the DNase molecules according to thepresent invention.

In certain further embodiments capillary electrophoresis may be used forthe separation of complex mixtures. For example a capillary may befilled with a suitable separation medium such as e.g. polyacrylamide andthe sample is put on one end (depending on the positioning of thecapillary e.g. the top) of the capillary. Depending on buffer and gelconditions the proteins and or peptides in the sample may be separatedby mass or charge, respectively.

Furthermore liquid chromatography may be applied for separating nucleicacids, proteins and/or peptides. Macromolecules such as nucleic acids,peptides and/or proteins may be separated according to their physicaland or chemical behaviour and or biological behaviour and orcombinations of these depending on the chromatographic media and orsolvents used for chromatography. In one embodiment the complex mixtureof proteins and/or peptides may be bound to a solid phase according totheir charge and eluted separately by increasing salt concentrations. Inone embodiment the complex mixture is separated by a solid phaseaccording to their mass by using a solid phase with a defined pore sizedistribution where proteins and or peptides enlarge their flow rate bydiffusion into the pores according to their mass.

In one embodiment the complex mixture of proteins and or peptides isseparated by a solid phase according to their shape by using a solidphase with a defined pore shape and or pore size distribution whereproteins and or peptides enlarge their flow rate by diffusion into thepores according to their shape. In one embodiment the complex mixture ofproteins and or peptides is bound to a solid phase according to theirhydrophobicity and eluted separately by applying a gradient ofhydrophobic solvents. In one embodiment one or all chromatographicmethods mentioned above combined in a manner which is suitable forseparating complex mixtures of proteins and or peptides.

In certain embodiments two dimensional HPLC may be used for separationof nucleic acids, proteins and/or peptides. For example a separation bymeans of ion exchange columns may be applied in combination with areversed phase column. The ion exchange column may for example be ananion or cation exchange column of a suitable strength for use in themethod disclosed herein. Materials for use in these HPLC methods areknown to those of ordinary skill in the art. In certain embodiments theeluates from the first column (eluted e.g. by means of increasing saltsteps) may be loaded onto the reversed phase column. The reversed phasecolumn may e.g. be eluted by an ascending gradient of an appropriatesolvent (e.g. acetonitril).

In certain embodiments of the present invention a pre-treatment of theseparated macromolecules such as e.g. proteins or polypeptides may beapplied prior to the detection reaction. Such procedures may for exampleemploy reduction or oxidation of proteins or peptides, proteolyticcleavage, modification of the proteins or peptides, derivatizaton orapplication of protecting moieties to prevent reactive parts of thepeptides from unwanted reactions. For example in certain embodimentssulfhydryl-groups may be prevented from oxidation. Generally a proteinextract for use in the methods according to the present invention maybut need not be digested with suitable enzymes such as trypsin or anyother protease to prepare peptides suitable for detection.

In certain embodiments of the present invention one or more fragmentsmay be present without subjecting the sample to any steps of samplepreparation. This may be due to activity of (digestive) proteolyticenzymes within the sample such as e.g. in stool, in fluids of thegastrointestinal tract or in gastrointestinal secretions. The fragmentsmay be detected by any means described herein. In one embodimentfragments may be detected in the course of a mass spectrometricanalysis. Detection of the respective fragment peaks corresponding tothe peptides derived from DNase may be especially useful for thedetection of the presence or absence and/or the level of DNase proteinsin samples.

In one embodiment of the present invention nucleic acids may but notneed to be subjected to a purification process prior to a subsequentdetection or amplification reaction. This may be desirable e.g. tofurther enhanced signal to noise ratios. Furthermore purification ofnucleic acids may be also applied subsequently to amplification reactionas the case may be.

Purification techniques for the purpose of purification of nucleic addsare known to those of ordinary skill in the art and comprise for examplegel electrophoresis, chromatography, precipitation, ultra centrifugationetc. E.g. the nucleic acids may be purified using electrophoresis in asuitable solid, viscous or liquid medium, such as a gel made fromsubstances known to those of skill in the art (agarose, polyacrylamid,starch etc.).

Alternatively nucleic acids may be purified using hybridisation of thenucleic acids to complementary or reverse-complementary nucleic acidprobes (e.g. fixed to a solid phase such as beads, membranes, slidesetc.) in the procedure of an affinity chromatography or other suitablecapture formats. Additionally precipitation methods for precipitation ofnucleic acids (e.g. using ethanol, isopropanol or other alcohol inappropriate concentration, trichloroacetic acid, or other suitable acidsor any other agent suitable for precipitation of nucleic acids fromsolutions) may be applied for the purification as well aschromatographic methods, such as Ion exchange chromatography, affinitychromatography etc. According to the present invention nucleic acids maybe purified using ultra centrifugation techniques, such as densitygradient centrifugation in e.g. an isokinetic or isopyknic manner orother suitable centrifugation techniques.

Generally a method for detection of the level of the marker moleculesfor use in the methods according to the present invention is any method,which is suited to detect and identify biological macromolecules such asnucleic acids, peptides and protein molecules in samples. In certainembodiments of the invention these methods may be methods exhibitinghigh sensitivity, so that even small amounts of molecules may bedetected. In further embodiments of the present invention standarddetection methods exhibiting suitable sensitivities may be employed. Anymethod may be employed such as e.g. those including detection reactionsin solution, methods employing solid phase adsorbed or coupled agents,etc. The method may be in vitro methods or may be methods to be appliedin vivo e.g. in the course of in vivo imaging procedures.

In certain embodiments more than one peptide derived from DNasepolypeptides and/or polynucleotides will be determined in a detectionprocedure. The detection of the level of marker polypeptides orfragments thereof according to the present invention may be thedetection of the level of single marker molecules in separated reactionmixtures as well as the detection of a combination of markerssimultaneously.

Furthermore the detection of the DNase nucleic acids, polypeptidesand/or polynucleotides as disclosed herein may be carded out incombination to one or more further detection reactions. These detectionreaction may for example be reaction for determination of the presenceof further suitable marker nucleic acids and/or polypeptides or of oneor more nucleic acids marker molecules in the samples. Further markermolecules, that may be suitable for the detection of proliferativedisorders in the course of a method as disclosed herein may comprisee.g. cyclins (Cyclin A, Cyclin, B, Cyclin E), cyclin-dependent kinaseinhibitors (p13.5, p14, p15, p16, p18, p19, p21, p27 etc.),cyclin-dependent kinases (cdk2, cdk4, cdk6 etc.) cell cycle regulatoryproteins (p14ARF, pRb, mdm2, p53), proliferation marker molecules (mcm2,mcm3, mcm4, mcm5, mcm6, mcm7, cdc2, cdc6, Ki67, Ki-S2, PCNA, DNApolymerase delta, rF Kappa B, etc.), marker for viral infection (such asHBV, HPV (especially high risk HPV: 16, 18, 31, 33, 38, 44, 45, 58, 68,etc.), HIV etc.) or other tumour marker proteins or nucleic acids (e.g.her2neu, CEA, PSA etc.).

In certain embodiments the detection of the level of DNase molecules isperformed indirectly by determination of the enzyme activity of theDNase. Enzyme activity may e.g. be determined by measuring degradationof enzyme substrates such as DNA. Methods for detection of DNaseactivities are known to those of skill in the art.

The detection of one or more molecular markers may be performed in asingle reaction mixture or in two or separate reaction mixtures. Thedetection reactions for several marker molecules may for example beperformed simultaneously in multi-well reaction vessels. The DNasenucleic acids and/or polypeptides disclosed herein may be detected usingmethods and/or reagents that specifically detect these molecules.Simultaneously one or more further markers may be detected using methodsand/or reagents that specifically detect them. The detection procedurefor each single marker may comprise one or more steps. In certainembodiments the detection procedure may comprise detection of the markermolecules by a primary detecting step followed by a further secondarydetecting step making the result of the procedure available forquantitative and/or qualitative analysis. Examples of detectionprocedures involving multiple steps may e.g. comprise the use of primaryand secondary and further binding agents.

In certain embodiments the detection procedure further may comprise areporter reaction indicating the level of the inventive DNasepolypeptides and/or nucleic acids. The reporter reaction may be forexample a reaction producing a coloured compound, a bioluminescence orchemiluminescence reaction, a fluorescence reaction, generally aradiation emitting reaction, or a reaction involving a chemical bindingreaction such as biotin binding or metal chelate binding.

In certain embodiments of the present invention procedures for thedetection reaction according to the present invention may employ forexample any immunological methods for detection of molecules, such asfor example Western blot, dot blot, immuno-precipitation orimmunological assays, such as ELISA, RIA, lateral flow assays etc.

In these embodiments determination of the (DNase) marker nucleic acidsand/or polypeptides may for example be carried out in a reactioncomprising a binding agent specific for the detection of the markermolecules. These binding agents may comprise for example nucleic acidprobes, antibodies and antigen-binding fragments, bifunctional hybridantibodies, peptidomimetics containing minimal antigen-binding epitopesetc. The binding agents may be used in many different detectiontechniques for example in southern-, northern-, western-blot, ELISA,lateral flow assay, (hybrid) capture assay, latex-agglutination,immuno-chromatographic strips or immuno-precipitation. Generally bindingagent based detection may be carried out as well in vitro as directly insitu for example in the course of an immuno-cytochemical stainingreaction. Any other method suitable for determining the amount ofparticular polypeptides in solutions of biological samples, such asbiochemical, chemical, physical or physico-chemical methods, may be usedaccording to the present invention.

Methods for detection of methylation of nucleic acids are known to thoseof skill in the art and may comprise for example methods employingchemical pretreatment of nucleic acids with e.g. sodium bisulphite,permanganate or hydrazine, and subsequent detection of the modificationby means of specific restriction endonucleases or by means of specificprobes e.g. in the course of an amplification reaction. The detection ofmethylation may furthermore be performed using methylation specificrestriction endonucleases.

In one embodiment of the invention the detection of the level of markermolecules is carried out by detection of the level of nucleic acidscoding for the marker molecules or fragments thereof present in thesample. The means for detection of nucleic acid molecules are known tothose skilled in the art. The procedure for the detection of nucleicacids can for example be carried out by a binding reaction of themolecule to be detected to complementary nucleic acid probes, proteinswith binding specificity for the nucleic acids or any other entitiesspecifically recognising and binding to said nucleic acids. This methodcan be performed as well in vitro as directly in situ for example in thecourse of a detecting staining reaction. Another way of detecting themarker molecules in a sample on the level of nucleic acids performed inthe method according to the present invention is an amplificationreaction of nucleic acids, which can be carried out in a quantitativemanner such as for example PCR, LCR or NASBA.

In certain embodiments of the present invention amplification ofribonucleic acids or of deoxy-ribonucleic acids may be applied to detectsmall amounts of DNase marker molecules or of small amounts of cellsexpressing DNase marker molecules in samples. This may be especiallyuseful for the detection of dispersed tumour cells in samples, or fordetection of DNase molecules, that have been dispersed to body fluidsfrom tumour cells, that expressed these DNase molecules. Generally thedetection of metastases, minimal residual disease or disseminated tumourcells in body samples may comprise a nucleic acid amplification reactionas mentioned above.

In the course of the detection of minimal residual disease generallydetection of DNase molecules such as peptides, proteins, DNA or mRNA inblood samples or the detection of disseminated cells may be suitable. Inthe course of the detection of DNase molecules an amplification reaction(e.g. PCR, LCR, NASBA) may be employed. In the course of the detectionof disseminated tumour cells may be separated from a body liquid andafter lysing of the cells DNase molecules may be detected in the lysate.In certain embodiments of the present invention the detection of thedispersed tumour cells may be carried out in lymph node samples or inbone marrow samples to detect metastases or disseminated tumour cells,that have spread to the respective samples. It must be understood, thatin the course of a detection of disseminated tumour cells any sampleobtainable from an individual may be useful.

In one embodiment of the present invention the detection of a carcinomasor their precursor lesions or the detection of metastases or minimalresidual disease in an individual may comprise the determination of theaccessibility of a specific region of a DNase molecule in samples. Thismay comprise e.g. the detection of the capability of a site specificbinding agent to react with DNase in a sample. Further more thedetection of carcinomas and their precursor lesions as well as thedetection of minimal residual disease or metastases may comprise thedetermination of the subcellular localization of DNase in cells.

Alternatively for the purpose of detection of disseminated tumour cells,metastases or minimal residual disease mass spectrometric detection ofnucleic acids may be applied subsequently to amplification, or may beapplied without amplification reaction.

In another embodiment of the invention the detection of the level ofmarker molecules is carried out by determining the level of expressionof a protein. The determination of the marker molecules on the proteinlevel may for example be carried out in a reaction comprising a bindingagent specific for the detection of the marker molecules. These bindingagents may comprise for example antibodies and antigen-bindingfragments, bifunctional hybrid antibodies, peptidomimetics containingminimal antigen-binding epitopes etc. The binding agents may be used inmany different detection techniques for example in western-blot, ELISA,lateral flow assay, latex-agglutination, immuno-chromatographic stripsor immuno-precipitation. Generally binding agent based detection may becarried out as well in vitro as directly in situ for example in thecourse of an immuno-cytochemical staining reaction. Any other methodsuitable for determining the amount of particular polypeptides insolutions of biological samples, such as biochemical, chemical, physicalor physico-chemical methods, can be used according to the presentinvention.

In certain embodiments of the present invention mass spectrometry may beused for detection of the DNase marker nucleic acids and/orpolypeptides. Generally any type of mass spectrometry may be used in themethod according to the present invention. The molecules to be analysedmay be ionised by any suitable method. In one embodiment the ionisationmethod in the course of mass spectrometry may be a matrix assisted laserdesorption ionisation, fast atom bombardment ionisaton, electron sprayionisaton or any other suitable method. Any technique known in the artfor mass spectrometric resolution and detection of the generated ionsmay be used in the method according to the present invention. The massspectrometric analysis may for example be performed by means of a timeof flight analyser, may employ an ion trap, a quadrupole, a sector fieldanalyser, a cyclotron etc.

The complete analysis including 2D-HPLC and mass spectrometricidentification may for example be carried out on theProteomeX-Workstation (ThermoFinnigan, San Jose, Calif., USA). Thesystem includes a HPLC system comprising two HPLC pumps and an autosampler which are connected to independently provide solvent to a strongcation exchange column and a reversed column. In the first step thetyptic digest is loaded onto the strong ion exchange column and washedusing a appropriate solvent to remove any contaminations not suitablefor further analysis. In increasing salt steps beginning with 1 mMammonium chloride and ending at 900 mM ammonium chloride fractions ofthe proteolytic peptides are eluted from the strong cation exchangecolumn and loaded onto the reversed phase column. Using the second HPLCpump the peptides on the reversed phase column are eluted by anascending acetonitril gradient from 5% to 80% acetonibil in water afterwashing the bound peptdes to remove access salt and conditioning thepeptides for the subsequent mass spectrometric analysis. The peptideseluting from the reversed phase column are measured on line by use of aelectrospray ionisation ion trap mass spectrometer (DECA LCQ,ThermoFinnigan, San Jose, Calif., USA) which enables the direct analysisand fragmentation of eluted peptides. Each reversed phase run ismonitored continually by the ESI-MS and ESI-MS/MS spectra and saved forsubsequent protein identification with the SEQUEST software package.SEQUEST uses peptide fragmentation mass spectra retrieved during thedata dependent MSIMS process which generated fragment mass spectra ofeluted peptides. The SEQUEST algorithm links experimentally derivedfragment spectra with in silico generated fragment spectra fromdatabases and enables correlation of the experimentally derived spectrumto the appropriate database record which identifies the peptide matchingto this record.

The fragments detected during the MS analysis may be identified bycomparison of these fragments to the data obtained from a database.Using appropriate algorithms proteins may be identified according to thefragment data obtained from the MS analysis.

In one embodiment peptide fragments obtainable by proteolytic cleavageof DNase proteins may be especially useful for the detection of DNaseproteins in samples. Detection of the presence or the level of DNaseproteins in samples may in one embodiment of the present inventioncomprise the detection of the presence or absence and/or the level ofone or more proteolytic fragment peptides derived from DNase proteins ina sample. In one embodiment the detection may comprise the detection ofthe respective fragment peaks in a mass spectrum or in a complex patternof different peptide fragment signals obtainable by a suitableanalytical method.

In certain embodiments the separation of the proteins, subsequentanalysis of the proteins and peptides and final identification of theproteins according to the detected mass spectra may be carried out in anassembled process.

Another technique suitable for peptide identification in complex samplesuses 2D-Electrophoresis instead of 2D liquid chromatography. Gel spotsstained with coomassie brilliant blue can be cut from the gel anddigested using trypsin. The subsequent identification of both singlepeptides as well as the “mass fingerprint” of the digested protein canbe performed using matrix assisted laser desorption and ionisaton massspectrometry or electrospray ionisaton mass spectrometry.

For detection In mass spectrometry of nucleic acids purified nucleicacids may be subjected to amplification reactions. Suitableamplification reactions are known to those of ordinary skill in the artand may comprise DNA based amplification as well as RNA basedamplification. Amplification reactions according to the presentinvention may comprise PCR, LCR, NASBA, etc. The amplification reactionmay be performed using one or more specific primers. In one embodimentof the invention an amplification reaction comprises the amplificationof a single nucleic acid. In another embodiment of the invention theamplification is carried out as a multiplex amplification reactionsimultaneously amplifying a set of several nucleic acids.

In one embodiment of the present invention the amplified nucleic acidsmay be used for a subsequent primer extension reaction, with or withoutprior purification which gives rise to nucleic acid fragments of 10 toabout 50 bp length.

In certain embodiments of the present invention immunological entitiesdirected against DNase may be detected. This detection reaction may becarried out in the course of detection of disorders associated with theexpression of DNase molecules or in the course of a immunotherapy fordetermination of the immuno-status of an individual or for monitoring ofthe effect of an immunization or vaccination therapy.

In one embodiment of the invention the detection of the level ofimmunological entities specific for DNase peptides is carried out on thelevel of antibodies. The method for detection of disorders according tothe present invention thus may employ the detection of immunologicalentities directed against one single peptide or the detection of a setof immunological entities. The use of a multiplicity of potentialpeptides raises the probability to detect the presence of a particulardisorder, and may furthermore give additional information useful instratification of a disorder, in monitoring the disease course or inassessment of prognosis concerning the disease course.

Immunological entities as used in the context of the present inventionshall comprise any components of the mammalian immune system, that areable to specifically react with an antigenic epitope. Such immunologicalentities may comprise for example antibodies, all immuno-globulins, suchas e.g. IgG, IgM, IgA, IgE, IgD, specific CD8+ T-cells or specificT-helper cells.

In this embodiment the detection may be e.g. performed using thespecific interaction between the respective DNase peptides with theantibodies. The determination of the presence or absence and/or thelevel of antibodies directed against DNase peptides in an individual mayfor example be carried out with recombinantly produced DNase peptides.The peptides can be used in many different detection techniques forexample in western-blot, ELISA or immuno-precipitation. In oneembodiment the detection of antibodies is carried out as antibodycapture assay (Antibodies A laboratory Manual, Harlow, Ed. et al., ColdSpring Harbor Laboratory 1988).

In another embodiment of the invention the detection of the specificantibodies is carried out using monoclonal or polyclonal antibodiesspecifically recognizing the antigen binding epitope of the firstantibodies. For this purpose the above mentioned immunological detectionprocedures may be applied. In a further embodiment chimeric antigens maybe employed in the detection reaction. Such chimeric antigens may forexample comprise fusion proteins combining the antigenic epitope of atumour associated polypeptide, recognized by the antibody in question,fused to another antigen, that may be recognized by a detectionantibody. The particular antigens within the chimeric polypeptide may beseparated by a linker or spacer region.

Any other method for determining the amount of particular antibodies orimmuno-globulins in biological samples can be used according to thepresent invention.

Generally the detection of the antibodies according to the presentinvention may be performed as well in vitro as directly in situ forexample in the course of an immuno-histochemical or immuno-cytochemicalstaining reaction.

In one embodiment of the present invention the immunological entitiesdirected against DNase molecules may be detected in a skin test. In thistesting format peptides of DNase may be introduced intradermally intothe skin of individuals in vivo. The testing format is known to those ofskill in the art from the so called TINE test or the SERO test stamp bySero-Mérieux. In this test the presence of immunological entitiesdirected against DNase molecules is diagnosed from a reaction of theindividual visible e.g. as inflammation of the skin at the respectivepoint of injection of the peptide. The evaluation thus relies onreddening of the skin and e.g. formation of reddish papules etc. on theskin. The test result may be dependent e.g. on the diameter of thereddening or the papules detectable after application of the antigens.The test result may be recorded e.g. by photo-documentation.

The peptides (also called In this context “antigens”) applicable for thedetection of immunological entities in individuals may be produced byany method known to those of ordinary skill in the art and may comprisefor example chemical synthesis of the polypeptides (fmoc-synthesis orequivalent) or may be produced recombinantly in any suitable host.However it must be obeyed, that the peptides are free of immunogeniccomponents other than the respective DNase derived peptides.

The amounts of peptides that must be applied in this testing format inorder to render visible immuno-reaction ranges between 0.1 μg and 10 μgof the purified peptide. In certain embodiments 0.05 μg, 0.1 μg, 0.5 μg,1 μg or 5 μg of the antigen or any value in between may be used perapplication. The antigens (peptides) are preferably applied as solution(whereas any other suitable format of application such as powder,aerosol or the like may be used according to the present invention aswell). The solvent may be any medically acceptable solution beingsterile and free of pyrogens, that does not cause inflammatory orimmunogenic reaction, when applied in a testing format as used in thepresented skin test

Cells exhibiting specificity for a DNase antigen may be detected by anymethods suitable for that purpose known to those of ordinary skill inthe art Methods may for example comprise proliferation-assays,cytokine-ELISAs, ELISpot assays, intracellular FACS-staining,PCR-mediated identification of peptide-specific cytokine (or similar)-expressing cells, tetramer-staining, cytotoxicity assays andDTH-(delayed type hypersensitivity) reactions.

In case of proliferation-assays induction of peptide-specific T-cellproliferation may be measured by methods known to those of skill in theart. This can be achieved by simple counting of cells, by measuringincorporation of labelled nucleotides into cellular DNA or by measuringlevel and/or activity of cellular protein(s). Cytokine-ELISA maycomprise identification of peptide-specific cytokine-secreting cells bymeasuring cytokine levels in supernatant In the course of an ELISpotassay the number of peptide-specific cytokine (i.e. IFN-g)—secretingcells in a sample is determined. Similarly the intracellularFACS-staining identifies cytokine-expressing cells on the protein level.In contrast (real-time) PCR may be used for identification ofpeptide-specific cytokine (or similar)—expressing cells on thetranscript level. In the course of a tetramer-staining assay the labelis a tetramer-molecule of recombinant MHC-class I molecules, loaded withspecific peptide and coupled to a dye. The tetramer binds to the T-cellreceptor. Cytotoxicity assays are a method for identification of cells,that can recognize and kill target cells in a peptide-specific manner.DTH-(delayed type hypersensitivity) reaction is based on the measuringof skin-reaction of vaccinated persons after Intradermal (or similar)application of peptide(s).

The method for the detection of immunological entities as disclosedherein may be performed for the purpose of monitoring In the course ofimmuno-therapeutic treatments of individuals. In this respect thepresence or absence and or the level of antibodies directed against aninventive peptide in an individual is determined. The determination ofthe level may be performed using the methods as se forth above. Thedetection may be performed at several consecutive points in time as tomonitor the timely alteration of the level of the immunologicalentities. The determination may for example be performed daily, weekly,monthly, once a year, or in once in a decade or at any interval inbetween.

In one preferred embodiment of the invention the level of markers issignificantly elevated compared to a non tumourous test sample. In thiscase the marker is over expressed in the sample. In another preferredembodiment of the present invention the level of the marker is loweredcompared to a non tumourous test sample. In a third embodiment there isno detectable expression of the marker at all in the test sample unlikein a control sample. In yet another embodiment there is detectable levelof non wild-type marker molecules. Non wild-type marker molecules maycomprise any marker molecules that deviate in sequence or structure fromthe structure or sequence, that is functional in wild type tissue notaffected by a cell proliferative disease. Wild type sequences orstructures are the sequences or structures predominantly present innormal cells or tissues. In one preferred embodiment of the inventionthe level of particular splicing variants of the marker gene is alteredin the test samples compared to the wild type issue. This may lead toaltered levels of splicing variants, new splicing variants,neo-peptides, altered ratios of different splicing variants of genes.

The detection procedure according to the present invention mayfurthermore comprise a cytochemical staining procedure rendering achromogenic or fluorescent staining of cells or cell compartments. Suchstaining procedures are known to those of skill in the art and may forexample comprise e.g. staining for acidophilic or basophilic structures,of sub cellular regions (e.g. the nucleus, the mitochondria, the Golgi,the cytoplasm etc.), of specific molecules (the chromosomes, of lipids,of glycoproteins, of polysaccharides etc.) in the cytological specimens.Fluorescence dyes such as DAPI, Quinacrin, Chromomycin, etc. may beemployed. Furthermore chromogenic dyes such as Azan, Acridin-orange,Hematoxylin, Eosin, Sudan-red, Thiazin—stains (Toluidin-blue, Thionin)may be applied. In other embodiments staining procedures such asPap-staining, Giemsa-staining, Hematoxylin-Eosin staining, van-Giesonstaining, Schiff-staining (using Schiff reagent), Feulgen staining,staining procedures employing precipitation of metals (such as e.g. ofsilver in staining procedures employing Silver Nitrate) or insolublestains such as e.g. of Tumbulls-blue (or other insoluble metalcyanides), etc. may be used in the course of a method as disclosedherein. It must be understood, that the named dyes and staining methodsshall be examples for the applicable methods and that any other methodknown in the art may be applied to a method as disclosed herein.

The staining procedures may produce chromogenic stains for lightmicroscopic inspection or fluorescent stains for inspection underfluorescence microscopic conditions. In another embodiment of thepresent invention radiation emitting procedures, procedures employingsubstances impairing the transmission of radiation or other contrastmedia for imaging of the cytological conditions in a sample (e.g. thegeneration of optical impression by means such as(micro-)autoradiographic or (mio-)radiographic picture generation) maybe of use for a method according to the present invention.

All the staining and imaging procedures may be used for analysis notonly in microscopic procedures but also in automated analysis proceduressuch flow cytometry, automated microscopic (computerized or computeraided) analysis or any other method for analysis of stained cytologicalspecimens.

The analysis of the staining or imaging results of the differentprocedures may be performed in a single analysis step or in differentsubsequent steps. E.g. the light microscopic inspection of a specimenmay be performed before or after fluorescence microscopic inspection ofthe specimen. In Fluorescence microscopy the analysis of differentstains with different excitation wavelengths may be analysessimultaneous or subsequently. Other imaging methods may be employedsimultaneously or subsequently to the named procedures.

There may be various circumstances, under which combinations ofdifferent staining methods will be suitable. E.g. in cases, where nosatisfying cytological staining results may be achieved byimmuno-chemical staining the additional application of generalcytological staining techniques may be suitable.

In certain embodiments of the present invention the method for detectionof the marker molecules in samples may be performed in an automatedmanner. The automation of the method may be achieved by automatedstaining and analysis of histological or cytological specimens on asolid surface by microscopic means. In another embodiment the automationmay comprise a flow-cytometric analysis of the staining of cells insolution.

In one preferred embodiment the detection of Issues expressing DNasegene products is carried out in form of molecular imaging procedures.The respective procedures are known to those of ordinary skill in theart. Imaging methods for use in the context of the present invention mayfor example comprise MRI, SPECT, PET and other methods suitable for invivo imaging.

In one embodiment the method may be based on the enzymatic conversion ofinert or labelled compounds to molecules detectable in the course ofmolecular imaging methods by the marker molecules. In another embodimentthe molecular imaging method may be based on the use of compoundscarrying a suitable label for in vivo molecular imaging, such as radioisotopes, metal ions etc., specifically binding to marker molecules invivo.

In a preferred embodiment of the invention these compounds are non-toxiccompounds and may be eliminated from the circulation of organisms, suchas humans, in a time span, that allows for performing the detection oflabel accumulated in tumour tissue over expressing the DNase markergene. In another preferred embodiment of the invention compounds areused for molecular imaging, for which clearance from the circulation isnot relevant for performing the molecular imaging reaction. This may befor example due to low background produced by the circulating moleculesetc. The compounds for use in molecular imaging methods are administeredin pharmaceutical acceptable form in compositions that may additionallycomprise any other suitable substances, such as e.g. otherdiagnostically useful substances, therapeutically useful substances,carrier substances or the like.

The DNase molecules disclosed according to the present invention may beused for diagnosis, monitoring of the disease course and prognosis incell proliferative disorders such as e.g. tumours.

Any methods for the detection of DNase molecules, of the accessibilityof regions on DNase molecules or of immunological entities directedagainst DNase molecules according to the present invention may e.g. beuseful in the course of diagnosis of carcinomas and their precursorlesions. Furthermore the methods for detection of DNase molecules or ofimmunological entities directed against DNase molecules may be used forthe determination of an immuno-status of individuals e.g. in the courseof immuno-therapy or vaccination procedures.

Diagnosis of carcinomas and their precursor lesions as used herein mayfor example comprise the detection of cells or tissues affected byabnormal growth. In one preferred embodiment diagnosis means the primarydetection of a disease in an organism or sample.

According to the present invention the method for diagnosis carcinomasand their precursor lesions may be applied In routine screening testsfor preventive aspects in order to detect said disease at an early stageof the onset of the disorder. For the purpose of early detection e.g.samples obtained by minimally invasive methods such as e.g. bloodsamples, stool samples, sputum samples, nipple aspirates, or samplesobtained by methods comprising colonoscopy, bronchioscopy,bronchioalveolar-lavage, ductal-lavage etc. may be employed. The methodsaccording to the present invention may be employed in the course of thedetection of early stages of tumours and of precursory lesions oftumours or cancers.

In another preferred embodiment the diagnostic method may be used todetermine the minimal residual disease of a tumour after primarytherapy. In this respect the method of the invention may be applied todetermine cells in body samples displaying abnormal expression of markermolecules according to the present invention, characteristic fortumours. Thus a spread of affected cells may be detected in body fluids.

In one embodiment of the invention the methods disclosed herein may beused for the detection and identification of metastases. The method maybe applied either for detection of metastases in body tissues or organsby the detection methods described herein, or the metastases may bediagnosed with respect to prognosis and prediction of disease course.

Monitoring of the disease course may comprise determining the levels ofmarker molecules at different time points, comparing the levels at thedifferent time points and assessing a diagnosis about the progression ofthe disease over the covered period of time. Thus monitoring may enablefor assessment of prognosis and/or for design of an adequate therapy fora particular patient.

Monitoring or diagnosis as used in the context of the present inventionmay also comprise the detection of an immuno-status of individuals inthe course of immuno therapy or vaccination therapy.

Prognosis of the disease course of a cell proliferative disorders suchas e.g. tumours according to the present invention may comprisedetermining the level of expression of one or more marker molecules,comparing the levels with data from subsequent studies in a database andprognosticating the disease course from said comparison. In a preferredembodiment the method may comprise the detection of the levels of a setof marker molecules, the distinct levels of which may characterizedistinct stages in the course of the disease. In a further embodiment ofthe invention the combination of the levels of a combination of markersmay be an indicator for the prognosis of the further disease course andmay build the basis for design of an adequate therapy.

Another aspect of the present invention is to provide a method fortherapy and/or vaccination. According to the present invention a therapyof cell proliferative disorders can be carried out using the inventiveDNase polypeptides and/or polynucleotides. The therapy may be forexample immuno-therapy or somatic gene therapy.

The inventive DNase polypeptides and/or polynucleotides may according tothe present invention be used for vaccination against cell proliferativedisorders. Vaccination according to the present invention may compriseadministering an immunogenic compound to an individual for the purposeof stimulating an immune response directed against said-immunogeniccompound and thus immunizing said individual against said immunogeniccompound. Stimulating an immune response may comprise inducing theproduction of antibodies against said compound as well as stimulatingcytotoxic T-cells. For the purpose of vaccination the polypeptides,nucleic acids and binding agents according to the present invention maybe administered in a physiological acceptable form. The composition tobe administered to individuals may comprise one or more antigeniccomponents, physiologically acceptable carrier substances or buffersolutions, immuno-stimulants and/or adjuvants. Adjuvants may comprisefor example Freund's incomplete adjuvant or Freund's complete adjuvantor other adjuvants known to those of skill in the art.

The composition may be administered in any applicable way such as e.g.intravenous, subcutaneous, intramuscular etc. The dosage of thecomposition depends on the particular case and purpose of thevaccination. It has to be adapted to parameters by the individualtreated such as age, weight, sex etc. Furthermore the type of the immuneresponse to be elicited has to be taken into account. In general it maybe preferable if an individual receives 100 μg-1 g of a polypeptideaccording to the present invention or 10⁶-10¹² MOI of a recombinantnucleic acid, containing a nucleic acid according to the presentinvention in a form that may be expressed in situ.

Individuals for the purpose of vaccination may be any organismscontaining the inventive tumour associated polypeptides and/orpolynucleotides and being able to get affected by cell proliferativedisorders.

Vaccination of individuals may be favourable e.g. in the case ofaltered, non wild-type sequences or structure of marker moleculesassociated with cell proliferative disorders. In one embodiment of theinvention vaccination may be applied in cases, where non-wild typequaternary structures of DNases appear in carcinomas and their precursorlesions, that are not present in wild type tissue.

Polypeptides disclosed herein may also be employed in adoptiveimmuno-therapy for the treatment of cancer. Adoptive immunotherapy maybe broadly classified into either active or passive immuno-therapy. Inactive immuno-therapy, treatment relies on the in vivo stimulation ofthe endogenous host immune system to react against tumours with theadministration of immune response-modifying agents (for example, tumourvaccines, bacterial adjuvants, and/or cytokines).

In passive immuno-therapy, treatment involves the delivery of biologicreagents with established tumour-immune reactivity (such as effectorcells or antibodies) that can directly or indirectly mediate anti-tumoureffects and does not necessarily depend on an intact host immune system.Examples of effector cells include T lymphocytes (for example, CD8+cytotoxic T-lymphocyte, CD4+ T-helper, tumour-infiltrating lymphocytes),killer cells (such as Natural Killer cells, lymphokine-activated killercells), B cells, or antigen presenting cells (such as dendritc cells andmacrophages) expressing the disclosed antigens. The polypeptidesdisclosed herein may also be used to generate antibodies oranti-idiotypic antibodies (as in U.S. Pat. No. 4,918,164), for passiveimmuno-therapy.

The predominant method of procuring adequate numbers of T-cells foradoptive immuno-therapy is to grow immune T-cells in vitro. Cultureconditions for expanding single antigen-specific T-cells to severalbillion in number with retention of antigen recognition in vivo are wellknown in the art These in vitro culture conditions typically utilizeintermittent stimulation with antigen, often in the presence ofcytokines, such as IL-2, and non-dividing feeder cells. As noted above,the immuno-reactive polypeptides described herein may be used to rapidlyexpand antigen-specific T cell cultures in order to generate sufficientnumber of cells for immuno-therapy. In particular, antigen-presentingcells, such as dendritic, macrophage or B-cells, may be pulsed withimmuno-reactive polypeptides or transfected with a nucleic acidsequence(s), using standard techniques well known in the art. Forexample, antigen presenting cells may be transfected with a nucleic acidsequence, wherein said sequence contains a promoter region appropriatefor increasing expression, and can be expressed as part of a recombinantvirus or other expression system. For cultured T-cells to be effectivein therapy, the cultured T-cells must be able to grow and distributewidely and to survive long term in vivo. Studies have demonstrated thatcultured T-cells can be induced to grow in vivo and to survive long termin substantial numbers by repeated stimulation with antigen supplementedwith IL-2 (see, for example, Cheever, M., et al, “Therapy With CulturedT Cells: Principles Revisited,” Immunological Reviews, 157:177, 1997).

The DNase polypeptides used herein may also be employed to generateand/or isolate tumour-reactive T-cells, which can then be administeredto the patent In one technique, antigen-specific T-cell lines may begenerated by in vivo immunization with short peptides corresponding toimmunogenic portions of the disclosed polypeptides. The resultingantigen specific CD8⁺ CTL clones may be isolated from the patient,expanded using standard tissue culture techniques, and returned to thepatient.

Alternatively, peptides corresponding to immunogenic portions of theDNase polypeptides used according to the invention may be employed togenerate tumour reactive T-cell subsets by selective in vitro simulationand expansion of autologous T-cells to provide antigen-specific T-cellswhich may be subsequently transferred to the patent as described, forexample, by Chang et al. (Crit. Rev. Oncol. Hematol., 22(3), 213, 1996).Cells of the immune system, such as T-cells, may be isolated from theperipheral blood of a patent using a commercially available cellseparation system, such as CellPro Incorporated's (Bothell, Wash.)CEPRATE.™. system (see U.S. Pat. No. 5,240,856; U.S. Pat. No. 5,215,926;WO 89/06280; WO 91/16116 and WO 92/07243). The separated cells arestimulated with one or more of the immuno-reactive polypeptidescontained within a delivery vehicle, such as a microsphere, to provideantigen-specific T-cells. The population of tumour antigen-specificT-cells is then expanded using standard techniques and the cells areadministered back to the patient.

In another embodiment, T-cell and/or antibody receptors specific for thepolypeptides can be cloned, expanded, and transferred into other vectorsor effector cells for use in adoptive immuno-therapy.

In a further embodiment, syngeneic or autologous dendritc cells may bepulsed with peptides corresponding to at least an immunogenic portion ofa polypeptide disclosed herein. The resulting antigen-specific dendritccells may either be transferred into a patient, or employed to stimulateT-cells to provide antigen-specific T-cells, which may, in turn, beadministered to a patient. The use of peptide-pulsed dendritic cells togenerate antigen-specific T-cells and the subsequent use of suchantigen-specific T-cells to eradicate tumours in a murine model has beendemonstrated by Cheever et al, Immunological Reviews, 157:177, 1997.

These carcinomas and their precursor lesions according to the presentinvention comprise conditions characterized by abnormal growthproperties of cells or tissues compared to the growth properties ofnormal control cells or tissues. The growth of the cells or tissues maybe for example abnormally accelerated or may be regulated abnormally.Abnormal regulation as used above may comprise any form of presence orabsence of non wild-type responses of the cells or tissues to naturallyoccurring growth regulating influences. The abnormalities in growth ofthe cells or tissues may be for example neoplastic or hyperplastic.

Disorders characterized by abnormal cell proliferation, as used in thecontext of the present invention, may comprise for example neoplasmssuch as benign and malignant tumours, carcinomas, sarcomas, leukemias,lymhomas or dysplasias. Tumours may comprise tumours of the head and theneck, tumours of the respiratory tract, tumours of the gastrointestinaltract, tumours of the urinary system, tumours of the reproductivesystem, tumours of the endocrine system, tumours of the central andperipheral nervous system, tumours of the skin and its appendages,tumours of the soft tissues and bones, tumours of the lymphopoietic andhematopoietic system, breast cancer, colorectal cancer, gastrointestinalcancer, anogenital cancer etc.

In certain embodiments the disorders are for example adenomas oradenocarcinomas of the colon, disorders of the respiratory tract such asSquamous Cell Lung Carcinoma, Small Cell Lung Carcinoma, Adenocarcinomaof the Lung, Large Cell Lung Carcinoma, Adeno-Squamous Lung Carcinoma,Cardnoid Tumour of the Lung, Broncheal Gland Tumour or (malignant)Mesothelioma, anogenital cancer such as, cervical cancer, vulval cancer,vaginal cancer, cancer of the rectum, cancer of the anus and cancer ofthe penis. In one embodiment the disorders may be breast cancer.

Additionally, vectors expressing DNase nucleic acids may be introducedInto stem cells taken from the patent and clonally propagated in vitrofor autologous transplant back into the same patent.

Monoclonal antibodies directed against DNase molecules be used astherapeutic compounds in order to diminish or eliminate tumours in amethod according to the present invention. The antibodies may be used ontheir own (for instance, to inhibit metastases) or coupled to one ormore therapeutic agents. Suitable agents in this regard include radionuclides, differentiation inducers, drugs, toxins, and derivativesthereof. Preferred radio nuclides include 90Y, 123I, 125I, 131I, 186Re,188Re, 211At, and 212Bi. Preferred drugs include methotrexate, andpyrimidine and purine analogues. Preferred differentiation inducersinclude phorbol esters and butyric acid. Preferred toxins include ricin,abrin, diptheria toxin, cholera toxin, gelonin, Pseudomonas exotoxin,Shigella toxin, and pokeweed antiviral protein.

In one embodiment of the invention the therapy of carcinomas and theirprecursor lesions may comprise the administration of antisense constructor ribozymes. The methods for administration of ribozymes or antisenseconstructs are known to those of skill in the art. The administrationmay take place as administration of naked nucleic acids or asadministration of nucleic acids that are suited for expression of therelevant active products in situ.

In another embodiment of the invention the treatment carcinomas andtheir precursor lesions may comprise the administration of bindingagents directed against the DNase polypeptides. These binding agents mayfor example be coupled to other compounds such as toxins, enzymes,radio-isotopes etc.

In another embodiment of the invention therapy of carcinomas and theirprecursor lesions may comprise the administration of antagonists oragonists of DNase polypeptides, of binding partners of the DNasepolypeptides, of inhibitors or enhancers of the expression of the DNasepolypeptides or of drugs identifiable by assays involving themeasurement of the activity of the DNase polypeptides. The methods foridentifying these substances are known to those of skill in the art

An example for a method for identifying a binding partner of a DNasepolypeptide (or related polypeptide) and/or polynucleotide may comprise:

-   -   (a) contacting the inventive DNase polypeptide of the invention        with a compound to be screened; and    -   (b) determining whether the compound effects an activity of the        polypeptide.

The DNase polypeptides may be used to screen for proteins or othercompounds that bind to the inventive colorectal lesion associatedpolypeptides or for proteins or other compounds to which the inventivecolorectal lesion associated polypeptide binds. The binding of the DNasepolypeptide and the molecule may activate (agonist), increase, inhibit(antagonist), or decrease activity of the DNase polypeptide or themolecule bound. Examples of such molecules include antibodies,oligonucleotides, proteins (e.g., receptors), or small molecules.

In one embodiment, the molecule is closely related to the natural ligandof the DNase polypeptide, e.g., a fragment of the ligand, or a naturalsubstrate, a ligand, a structural or functional mimetic; see, e.g.,Coligan, Current Protocols in Immunology 1(2) (1991); Chapter 5.Similarly, the molecule can be closely related to a natural receptor towhich the DNase might bind, or at least, a fragment of the receptorcapable of being bound by the DNase polypeptide (e.g., active site). Ineither case, the molecule can be rationally designed using knowntechniques.

Preferably, the screening for these molecules involves producingappropriate cells which express the DNase polypeptide, either as asecreted protein or on the cell membrane. Preferred cells include cellsfrom mammals, yeast, Drosophila, or E. coli. Cells expressing theinventive colorectal lesion associated polypeptide (or cell membranecontaining the expressed polypeptide) are then preferably contacted witha test compound potentially containing the molecule to observe binding,stimulation, or inhibition of activity of DNase polypeptide.

The assay may simply test binding of a candidate compound to the DNasepolypeptide, wherein binding is detected by a label, or in an assayinvolving competition with a labelled competitor. Further, the assay maytest whether the candidate compound results in a signal generated bybinding to the DNase polypeptide.

Alternatively, the assay can be carried out using cell-freepreparations, polypeptide/molecule affixed to a solid support, chemicallibraries, or natural product mixtures. The assay may also simplycomprise the steps of mixing a candidate compound with a solutioncontaining the DNase, measuring the DNase polypeptide/molecule activityor binding, and comparing the DNase polypeptide/molecule activity orbinding to a standard.

Preferably, an ELISA assay can measure the DNase level or activity in asample (e.g., biological sample) using a monoclonal or polyclonalantibody. The antibody can measure the DNase polypeptide level oractivity by either binding, directly or indirectly, to the DNasepolypeptide or by competing with the DNase polypeptide for a substrate.All of these above assays can be used to screen for diagnostic orprognostic markers and for therapeutic agents. The molecules discoveredusing these assays can be used to treat disease or to bring about aparticular result in a patient (e.g., elimination of a epithelial tumouror stop of progression of tumour growth) by activating or Inhibiting theDNase polypeptide molecules. Moreover, the assays can discover agentswhich may inhibit or enhance the production of the DNase polypeptidesfrom suitably manipulated cells or tissues.

Therefore, the invention includes a method of identifying compounds foruse in treatment of carcinomas and their precursor lesions which bind toa DNase polypeptides comprising the steps of: (a) incubating a candidatebinding compound with a DNase polypeptide; and (b) determining ifbinding has occurred.

Moreover, the invention includes a method of identifyingactivators/agonists or inhibitors/antagonists of the inventivecolorectal lesion associated polypeptide for use in treatment ofdisorders characterized by abnormal cell proliferation comprising thesteps of: (a) incubating a candidate compound with a DNase polypeptide;b) assaying a biological activity of the DNase (enzymatic activity orother), and (c) determining if a biological activity of the DNasepolypeptide has been altered.

In a further embodiment, the present invention relates to method ofidentifying and obtaining a drug candidate for therapy of carcinomas andtheir precursor lesions comprising the steps of

a. contacting a DNase or a cell expressing said DNase polypeptide in thepresence of components capable of providing a detectable signal inresponse

-   -   to altered regulation of cell proliferation    -   to altered activity of a DNase polypeptide    -   to altered cell differentiation,

with said drug candidate to be screened under conditions to allowprotein degradation, and

b. detecting presence or absence of a signal or increase of the signalgenerated from DNase polypeptide activity, cell proliferation ordifferantiation, wherein the presence or increase of the signal isindicative for a putative drug.

Experiments using animals or isolated cells or cell lines may be used toexamine the proliferative behaviour of cells or tissues in dependenceDNase polypeptide action. The same procedures may be employed for thestudy of cell differentiation.

The drug candidate may be a single compound or a plurality of compounds.The term “plurality of compounds” in a method of the invention is to beunderstood as a plurality of substances which may or may not beidentical.

Said compound or plurality of compounds may be chemically synthesized ormicrobiologically produced and/or comprised in, for example, samples,e.g., cell extracts from, e.g., plants, animals or micro organisms.Furthermore, said compound(s) may be known in the art but hitherto notknown to be capable of suppressing or activating a DNase polypeptide.The reaction mixture may be a cell free extract or may comprise a cellor tissue culture. Suitable set ups for the method of the Invention areknown to the person skilled in the art and are, for example, generallydescribed in Alberts et al., Molecular Biology of the Cell, thirdedition (1994) and in the appended examples. The plurality of compoundsmay be, e.g., added to the reaction mixture, culture medium, injectedinto a cell or otherwise applied to the transgenic animal. The cell orissue that may be employed in the method of the invention preferably isa host cell, mammalian cell or non-human transgenic animal of theinvention described in the embodiments hereinbefore.

If a sample containing a compound or a plurality of compounds isidentified in the method of the invention, then it is either possible toisolate the compound from the original sample identified as containingthe compound capable of suppressing or activating a DNase polypeptide,or one can further subdivide the original sample, for example, if itconsists of a plurality of different compounds, so as to reduce thenumber of different substances per sample and repeat the method with thesubdivisions of the original sample. Depending on the complexity of thesamples, the steps described above can be performed several times,preferably until the sample identified according to the method of theinvention only comprises a limited number of or only one substance(s).Preferably said sample comprises substances of similar chemical and/orphysical properties, and most preferably said substances are identical.

Several methods are known to the person skilled in the art for producingand screening large libraries to identify compounds having specificaffinity for a target. These methods include the phage-display method inwhich randomised peptides are displayed from phage and screened byaffinity chromatography to an immobilized receptor; see, e.g., WO91/17271, WO 92/01047, U.S. Pat. No. 5,223,409. In another approach,combinatorial libraries of polymers immobilized on a chip aresynthesized using photolithography; see, e.g., U.S. Pat. No. 5,143,854,WO 90/15070 and WO 92/10092. The immobilized polymers are contacted witha labelled receptor and scanned for label to identify polymers bindingto the receptor.

The synthesis and screening of peptide libraries on continuous cellulosemembrane supports that can be used for identifying binding ligands ofthe DNase polypeptide and thus possible inhibitors and activators isdescribed, for example, in Kramer, Methods Mol. Biol. 87 (1998), 25-39.This method can also be used, for example, for determining the bindingsites and the recognition motifs in the DNase polypeptides. In likemanner, the substrate specificity of the DnaK chaperon was determinedand the contact sites between human interleukin-6 and its receptor; seeRudiger, EMBO J. 16 (1997), 1501-1507 and Weiergraber, FEBS Lett. 379(1996), 122-126, respectively.

Furthermore, the above-mentioned methods can be used for theconstruction of binding super topes derived from the polypeptide of theinvention. A similar approach was successfully described for peptideantigens of the anti-p24 (HIV-1) monoclonal antibody; see Kramer, Cell91 (1997), 799-809. A general route to fingerprint analyses ofpeptide-antibody interactions using the clustered amino acid peptidelibrary was described in Kramer, Mol. Immunol. 32 (1995), 459-465. Inaddition, antagonists of the DNase can be derived and identified frommonoclonal antibodies that specifically react with the polypeptide ofthe invention in accordance with the methods as described in Doring,Mol. Immunol. 31 (1994), 1059-1067.

More recently, WO 98/25146 described further methods for screeninglibraries of complexes for compounds having a desired property,especially, the capacity to agonize, bind to, or antagonize a DNasepolypeptide or its cellular receptor. The complexes in such librariescomprise a compound under test, a tag recording at least one step insynthesis of the compound, and a tether susceptible to modification by areporter molecule. Modification of the tether is used to signify that acomplex contains a compound having a desired property. The tag can bedecoded to reveal at least one step in the synthesis of such a compound.Other methods for identifying compounds which interact with the DNasepolypeptides or DNase nucleic acid molecules encoding such moleculesare, for example, the in vitro screening with the phage display systemas well as filter binding assays or “real time” measuring of interactionusing, for example, the BIAcore apparatus (Pharmacia).

All these methods can be used in accordance with the present inventionto identify activators/agonists and inhibitors/antagonists of the DNasepolypeptide or related polypeptide for use in a method of the presentinvention.

Various sources for the basic structure of such an activator orinhibitor can be employed and comprise, for example, mimetic analoguesof the polypeptide of the invention. Mimetic analogues of the DNasepolypeptide of the invention or biologically active fragments thereofcan be generated by, for example, substituting the amino acids that areexpected to be essential for the biological activity with, e.g., stereoisomers, i.e. D-amino acids; see e.g., Tsukida, J. Med. Chem. 40 (1997),3534-3541. Furthermore, in case fragments are used for the design ofbiologically active analogues pro-mimetic components can be incorporatedinto a peptide to re-establish at least some of the conformationalproperties that may have been lost upon removal of part of the originalpolypeptide; see, e.g., Nachman, Regul. Pept. 57 (1995), 359-370.

Furthermore, the DNase polypeptide can be used to identify syntheticchemical peptide mimetic that bind to or can function as a ligand,substrate, binding partner or the receptor of the polypeptide of theinvention as effectively as does the natural polypeptide; see, e.g.,Engleman, J. Clin. Invest 99 (1997), 2284-2292. For example, foldingsimulations and computer redesign of structural motifs of thepolypeptide of the invention can be performed using appropriate computerprograms (Olszewski, Proteins 25 (1996), 286-299; Hoffman, Comput. Appl.Biosci. 11 (1995), 675-679). Computer modelling of protein folding canbe used for the conformational and energetic analysis of detailedpeptide and protein models (Monge, J. Mol. Biol. 247 (1995), 995-1012;Renouf, Adv. Exp. Med. Biol. 376 (1995), 37-45). In particular, theappropriate programs can be used for the identification of interactivesites of the DNase polypeptides and their possible ligand or otherinteracting proteins by computer assistant searches for complementarypeptide sequences (Fassina, Immunomethods 5 (1994), 114-120. Furtherappropriate computer systems for the design of protein and peptides aredescribed in the prior art, for example in Berry, Biochem. Soc. Trans.22 (1994), 1033-1036; Wodak, Ann. N.Y. Acad. Sci. 501 (1987), 1-13;Pabo, Biochemistry 25 (1986), 5987-5991.

The results obtained from the above-described computer analysis can beused for, e.g., the preparation of peptide mimetic of the DNase proteinor fragments thereof. Such pseudo-peptide analogues of the natural aminoacid sequence of the protein may very efficiently mimic the parentprotein (Benkirane, J. Biol. Chem. 271 (1996), 33218-33224). Forexample, incorporation of easily available archival-amino acid residuesinto a protein of the invention or a fragment thereof results in thesubstitution of amide bonds by polymethylene units of an aliphaticchain, thereby providing a convenient strategy for constructing apeptide mimetic (Banerjee, Biopolymers 39 (1996), 769-777).

Superactive peptidomimetic analogues of small peptide hormones in othersystems are described in the prior art (Zhang, Biochem. Biophys. Res.Commun. 224 (1996), 327-331). Appropriate peptide mimetics of theprotein of the present invention can also be identified by the synthesisof peptide mimetic combinatorial libraries through successive amidealkylation and testing the resulting compounds, e.g., for their bindingand immunological properties. Methods for the generation and use ofpeptidomimetic combinatorial libraries are described in the prior art,for example in Ostresh, Methods in Enzymology 267 (1996), 220-234 andDorner, Bioorg. Med. Chem. 4 (1996), 709-715.

Furthermore, a three-dimensional and/or crystallographic structure ofthe DNase polypeptide can be used for the design of peptide mimeticinhibitors of the biological activity of the polypeptide of theinvention (Rose, Biochemistry 35 (1996), 12933-12944; Rutenber, Bioorg.Med. Chem. 4 (1996), 1545-1558).

The structure-based design and synthesis of low-molecular-weightsynthetic molecules that mimic the activity of the native biologicalpolypeptide is further described in, e.g., Dowd, Nature Biotechnol. 16(1998), 190-195; Kieber-Emmons, Current Opinion Biotechnol. 8 (1997),435-441; Moore, Proc. West Pharmacol. Soc. 40 (1997), 115-119; Mathews,Proc. West Pharmacol. Soc. 40 (1997), 121-125; Mukhija, European J.Biochem. 254 (1998), 433-438.

It is also well known to the person skilled in the art, that it ispossible to design, synthesize and evaluate mimetics of small organiccompounds that, for example, can act as a substrate or ligand to theDNase polypeptides used in the invention or the related polypeptide. Forexample, it has been described that D-glucose mimetics of hapalosinexhibited similar efficiency as hapalosin in antagonizing multidrugresistance assistance-associated protein in cytotoxicity; see Dinh, J.Med. Chem. 41 (1998), 981-987.

The DNase nucleic acid molecule can also serve as a target foractivators and inhibitors. Activators may comprise, for example,proteins that bind to the mRNA of a gene encoding a DNase polypeptide,thereby stabilizing the native conformation of the mRNA and facilitatingtranscription and/or translation, e.g., in like manner as Tat proteinacts on HIV-RNA Furthermore, methods are described in the literature foridentifying nucleic acid molecules such as an RNA fragment that mimicsthe structure of a defined or undefined target RNA molecule to which acompound binds inside of a cell resulting in retardation of cell growthor cell death; see, e.g., WO 98/18947 and references cited therein.These nucleic acid molecules can be used for identifying unknowncompounds of pharmaceutical and/or agricultural interest, and foridentifying unknown RNA targets for use in treating a disease. Thesemethods and compositions can be used in screening for novel antibiotics,bacteriostatics, or modifications thereof or for identifying compoundsuseful to alter expression levels of proteins encoded by a nucleic acidmolecule.

Alternatively, for example, the conformational structure of the RNAfragment which mimics the binding site can be employed in rational drugdesign to modify known antibiotics to make them bind more avidly to thetarget. One such methodology is nuclear magnetic resonance (NMR), whichis useful to identify drug and RNA conformational structures. Stillother methods are, for example, the drug design methods as described inWO 95/35367, U.S. Pat. No. 5,322,933, where the crystal structure of theRNA fragment can be deduced and computer programs are utilized to designnovel binding compounds which can act as antibiotics.

Some genetic changes lead to altered protein conformational states. Forexample, some mutant the Inventive colorectal lesion associatedpolypeptides may possess a tertiary structure that renders them far lesscapable of protein degradation. Restoring the normal or regulatedconformation of mutated proteins is the most elegant and specific meansto correct these molecular defects, although it may be difficult Ofparticular Interest In this regard is the consensus domain of theInventive colorectal lesion associated polypeptide.

The compounds which can be tested and identified according to a methodsof the invention may be expression libraries, e.g., cDNA expressionlibraries, peptides, proteins, nucleic acids, antibodies, small organiccompounds, hormones, peptidomimetics, PNAs or the like (Milner, NatureMedicine 1 (1995), 879-880; Hupp, Cell 83 (1995), 237-245; Gibbs, Cell79 (1994), 193-198 and references cited supra). Furthermore, genesencoding a putative regulator of the DNase polypeptide and/or whichexert their effects up- or downstream the DNase polypeptide may beidentified using, for example, insertion mutagenesis using, for example,gene targeting vectors known in the art Said compounds can also befunctional derivatives or analogues of known inhibitors or activators.Such useful compounds can be for example transacting factors which bindto the inventive tumour associated polypeptide or regulatory sequencesof the gene encoding it. Identification of transacting factors can becarried out using standard methods in the art (see, e.g., Sambrook,supra, and Ausubel, supra).

To determine whether a protein binds to the DNase protein itself orregulatory sequences, standard native gel-shift analyses can be carriedout In order to identify a transacting factor which binds to the proteinor regulatory sequence, the protein or regulatory sequence can be usedas an affinity reagent in standard protein purification methods, or as aprobe for screening an expression library.

The identification of nucleic acid molecules which encode polypeptideswhich interact with the inventive DNase described above can also beachieved, for example, as described in Scofield (Science 274 (1996),2063-2065) by use of the so-called yeast “two-hybrid system”. In thissystem the polypeptide encoded by a nucleic acid molecule according tothe invention or a smaller part thereof Is linked to the DNA-bindingdomain of the GAL4 transcription factor. A yeast strain expressing thisfusion polypeptide and comprising a lacZ reporter gene driven by anappropriate promoter, which is recognised by the GAL4 transcriptionfactor, is transformed with a library of cDNAs which will express plantproteins or peptides thereof fused to an activation domain. Thus, If apeptide encoded by one of the cDNAs is able to interact with the fusionpeptide comprising a peptide of a inventive DNase polypeptide, thecomplex is able to direct expression of the reporter gene. In this waythe nucleic acid molecules according to the invention and the encodedpeptide can be used to identify peptides and proteins interacting withthe DNase protein. It is apparent to the person skilled in the art thatthis and similar systems may then further be exploited for theidentification of inhibitors of the binding of DNase proteins.

Once the transacting factor is identified, modulation of its binding toor regulation of expression of DNase polypeptide can be pursued,beginning with, for example, screening for inhibitors against thebinding of the transacting factor to the DNase protein of the presentinvention. Activation or repression of the inventive DNase proteinscould then be achieved in animals by applying the transacting factor (orits inhibitor) or the gene encoding it, e.g. in an expression vector. Inaddition, if the active form of the transacting factor is a dimer,dominant-negative mutants of the transacting factor could be made inorder to inhibit its activity.

Furthermore, upon identification of the transacting factor, furthercomponents in the pathway leading to activation (e.g. signaltransduction) or repression of a gene involved in the control of theinventive tumour associated polypeptide then can be identified.Modulation of the activities of these components can then be pursued, inorder to develop additional drugs and methods for modulating themetabolism of protein degradation in animals. Thus, the presentinvention also relates to the use of the two-hybrid system as definedabove for the identification of the inventive tumour associatedpolypeptide or activators or inhibitors of the inventive DNasepolypeptide.

The compounds isolated by the above methods also serve as lead compoundsfor the development of analogue compounds. The analogues should have astabilized electronic configuration and molecular conformation thatallows key functional groups to be presented to the DNase polypeptide orits possible receptor in substantially the same way as the leadcompound. In particular, the analogue compounds have spatial electronicproperties which are comparable to the binding region, but can besmaller molecules than the lead compound, frequently having a molecularweight below about 2 kD and preferably below about 1 kD.

Identification of analogue compounds can be performed through use oftechniques such as self-consistent field (SCF) analysis, configurationinteraction (CI) analysis, and normal mode dynamics analysis. Computerprograms for implementing these techniques are available; e.g., Rein,Computer-Assisted Modeling of Receptor-Ugand Interactions (Alan Liss,New York, 1989). Methods for the preparation of chemical derivatives andanalogues are well known to those skilled in the art and are describedin, for example, Beilstein, Handbook of Organic Chemistry, Springeredition New York Inc., 175 Fifth Avenue, New York, N.Y. 10010 U.S.A. andOrganic Synthesis, Wiley, N.Y., USA.

Furthermore, said derivatives and analogues can be tested for theireffects according to methods known in the art; see also supra.Furthermore, peptidomimetics and/or computer aided design of appropriatederivatives and analogues can be used, for example, according to themethods described above.

In a preferred embodiment of the above-described methods of theinvention said cell is a cell of or, obtained by a method of theinvention or is comprised in the above-described transgenic non-humananimal.

Once the described compound has been identified and obtained, it ispreferably provided in a therapeutically acceptable form.

It must be understood, that the compounds and methods as disclosedthroughout this text are applicable to any mammalian individual. Thusthe compounds and methods may be applied to animals as well as to humanbeings and are such useful in veterinary medical as in medical purposes.Animals, that may be of especial interest with respect to the presentinvention are companion animals, such as cats, dogs, etc. animals ofagricultural interest such as cows, pigs, horses, laboratory animalssuch as rats, mice, hamsters, rabbits etc. and any other animal, thatmay be affected by a disorder characterized by abnormal growth of cells.

The present invention provides compounds and methods useful fordetection and treatment carcinomas and their precursor lesions. In oneaspect the present invention provides a method for the detection ofcarcinomas and their precursor lesions based on the determination of thepresence or absence and/or the level of expression of DNase molecules inbiological samples. This detection method may e.g. be employed in thecourse of early detection of neoplasias and precursory stages oftumours. In a second aspect the present invention provides a method fortreatment of carcinomas and their precursor lesions by modulation ofDNase gene products as therapeutically active agents. The invention alsoprovides for therapeutic methods based on the modulation of the activityof DNase polypeptides. It is one aspect of the invention to provide amethod for rational tumour management based on the detection of DNasegene products in patent samples and the tailoring of a therapycorrelated to the detected over expression of said DNase gene products.Furthermore the present invention provides for a research or diagnostictest kit for performing the reactions involved in the detection of thepresence or absence and/or the level of over expression of DNase genes.Finally the present invention relates to pharmaceutical compositionsapplicable in the treatment of carcinomas and their precursor lesionscomprising DNase compounds as disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Immunohistological specimen stained with antibodies directedagainst DNase X; A: colon carcinoma; B: corresponding normal tissue; thecolon carcinoma specimen as well as the normal Issue has been subjectedto an immunochemical staining reaction employing a primary antibodydirected against DNase X; the figure shows nuclear positive staining forDNase X in the tumour cells; in normal tissue intraepithelial endocrinecells show immunoreactivity for DNase X in the cytoplasm; forexperimental details see Example 1.

FIG. 2: Immunohistological specimen stained with antibodies directedagainst DNase X; A: gastric carcinoma; B: corresponding normal tissue; agastric carcinoma specimen as well as corresponding normal tissue hasbeen subjected to an immunochemical staining reaction employing aprimary antibody directed against DNase X; the figure shows nuclearpositive staining for DNase X In the tumour cells; in normal tissueglandular endocrine cells show immunoreactivity for DNase X in thecytoplasm; for experimental details see Example 1.

FIG. 3: Immunohistological specimen stained with antibodies directedagainst DNase X; a lung carcinoma specimen has been subjected to animmunochemical staining reaction employing a primary antibody directedagainst DNase X; the figure shows nuclear positive staining for DNase Xin the tumour cells; for experimental details see Example 1.

FIG. 4: immunohistological specimen stained with antibodies directedagainst DNase X; A: adeno-carcinoma at the oesophago-gastical junction;B: corresponding normal oesophageal tissue; an oesophageal carcinomaspecimen as well as corresponding normal tissue has been subjected to animmunochemical staining reaction employing a primary antibody directedagainst DNase X; the figure shows nuclear positive staining for DNase Xin the tumour cells; no staining is visible in the normal tissue; forexperimental details see Example 1.

FIG. 5: Immunohistological specimen stained with antibodies directedagainst DNase X; a cervical dysplasia specimen (CINIII) has beensubjected to an immunochemical staining reaction employing a primaryantibody directed against DNase X; the figure shows nuclear positivestaining for DNase X in the tumour cells; for experimental details seeExample 1.

FIG. 6: Immunohistological specimen stained with antibodies directedagainst DNase X; a ductal carcinoma In situ has been subjected to animmunochemical staining reaction employing a primary antibody directedagainst DNase X; the figure shows nuclear positive staining for DNase Xin the tumour cells; the neighbouring normal issue shows no staining forDNase X; for experimental details see Example 1.

FIG. 7: Graph inversely showing DNase activities measured in serum ofindividuals with different kinds of carcinomas; the graph displays, thaton average all carcinomas tested except for rectal carcinomas areaccompanied by elevated serum DNase activity. (details in Example 4)

The following examples are given for the purpose of illustration onlyand are not intended to limit the scope of the invention disclosedherein.

EXAMPLE 1 Immunochemical Detection of the Overexpression of DNase X inTissue Samples of Carcinomas

Sections of formalin fixed, paraffin embedded tissue samples of thecolon were immunocytochemically stained using antibodies specific forDNase X.

The sections were rehydrated through incubation in xylene and gradedethanol, and transferred to Aqua bidest. Antigen Retrieval was carriedout with 10 mM citrate buffer (pH 6.0) Therefore the slides were heatedin a waterbath for 40 min at 95° C. The slides were cooled down to RTfor 20 minutes, transferred to washing buffer (PBS/0.1% Tween20).

For inactivation of endogenous peroxidase the samples are incubated with3% H₂O₂ for 20 min at RT and afterwards washed in PBS/0.1% Tween20 for 5to 10 min.

The slides were then incubated with the primary antibody, rat anti-DNaseX (1:25) (for 1 hour at RT, the slides were then rinsed with washingbuffer and placed in a fresh buffer bath for 5 min. The antibodyemployed is directed against the peptide sequence casltkkrldklelrtepgfof human DNase X.

Afterwards the slides were incubated with the secondary antibody (goatant rat (1:500)). Washing was performed 3 times for 5 minutes. Excessbuffer was tapped off and the specimen was covered with 100 μl ofvisualization reagent for 30 min at RT. Slides were washed as before,and covered with 200 μl substrate-chromogen solution (DAB) for 10 min.Then slides were washed as before and counterstained for 3 min in a bathof haematoxylin. Residual haematoxylin was rinsed with distilled water,and specimens were mounted and coverslipped with an aqueous mountingmedium.

The microscopic examination of the slides reveals, that cellsimmunoreactive with DNase X can be found in samples, that maymicroscopically be identified as samples of colorectal carcinoma. Incarcinomas the DNase X specific staining is concentrated in the cellularnucleus. In contrast in control samples very few single cells may bestained. In all non-cancerous cells the staining is cytoplasmic, whereasin cells of carcinomas and their precursor lesions the staining islocated nuclear. Especially in gastrointestinal issues endocrine cellsshow cytoplasmic staining for DNase X. No other cells ingastrointestinal issues may be stained. In other tested tissues there isno positive staining. In these cases the staining is localized in thecytoplasm of the cells.

The above described immunohistochemical staining procedure wasfurthermore applied to tissues from breast-, lung-, cervical-(CINIII),gastric-, oesophageal-, endometral-, ovarian-carcinomas. In all thesecases nuclear staining for DNase X could be observed in the cancerouscells. In normal tissue few to no staining was identified.

Moreover metastases from colorectal carcinoma located in the liver wereanalysed by immunochemical procedures as described above. The resultshowed nuclear staining in the tumour cells and no staining in thesurrounding normal tissue.

Immunochemical analysis of peripheral venous blood, of bone marrow andof lymphocytes by the described methods revealed no immunoreactivity forDNase X in samples obtained from normal control individuals. Thisindicates, that disseminated tumour cells that are immunoreactive withDNase X might be identified in these samples by specific immunochemicalstaining with antibodies directed against DNase X.

The results show, that the staining with reagents specific for DNase Xallows to identify carcinomas in biological samples. In carcinomas andtheir precursor lesions there is nuclear staining for DNase X with theemployed antibody, whereas in normal tissue only few cells may bestained in the cytoplasm.

Generally it may be stated that staining Is concentrated in the nucleusof the cells. However cytoplasmic immunoreactivity may also be seen intransformed cells (tumor tissue).

EXAMPLE 2 Detection of Disseminated Tumour Cells in Lymph Nodes ofIndividuals

Lymph node samples of patients obtained in the course of surgicalresection of adenocarcinomas of the colon were employed to determine thepresence of cells showing immunoreactivity with DNase X specific bindingagents. In total samples from 7 patients with colon-carcinomas wereincluded.

Immunohistochemical staining was performed as given in Example 1.

The experiment reveals that staining with the antibody directed againstDNase X may be detected in samples of patients with carcinoma.

An immuno-histochemical staining for DNase X in the samples couldimprove the detectability of the disseminated tumour cells in the lymphtissue.

EXAMPLE 3 Early Diagnosis of Ductal Carcinoma In Situ by Detection ofDNase X in Cells Contained in Ductal Lavage Fluid

A collective of 14 individuals was included in this study. 7 patientswere identified as having calcifications in breast ducts indicative ofearly stage ductal carcinoma in situ by mammographic examination. 7individuals did not show any signs indicating a neoplastic lesion of thebreast.

Ductal lavages were performed with all 14 individuals and cells wereisolated from the lavage fluid.

Cytological preparations were done from the ravages fluids by ThinPrep™technology. Immuno-chemical staining was performed as described inExample 1.

The experiment reveals the presence of cells immunoreactive for DNase Xin the cytological preparation of 9 individuals. In the samples of allpatents with a mammographic diagnosis Indicating the presence of ductalcarcinoma in situ, cells immunoreactive for DNase X could be identified.

The result shows, that the conventional methods for identification ofearly stages of neoplasias of the breast may be improved by methodsbased on the detection of the DNase X immunoreactivity presented herein.

EXAMPLE 4 Diagnosis of Gastrointestinal Carcinomas In Situ by Detectionof DNase Activity in Body Fluids

Using a monoclonal antibody directed against the DNaseX protein anoverexpression of the DNaseX protein has been detected in every tumorentity which was analysed by immunohistochemistry. Therefore theoverexpression of the DNaseX protein represents a general phenomen intumors. Since the DNaseX protein is a soluble protein with an intrinsicenzymatic activity, the overexpression found on immunohistochemistrylevel suggests that the overexpression of the DNaseX protein found intumor cells could lead to an enhanced level of DNaseX protein or DNaseactivity in body fluids of tumor patients.

To test this hypothesis we first analysed if the overexpression of theDNaseX protein leads to an enhanced DNase activity in sera of tumorpatients.

Serum from individuals with different carcinomas were used to find out,whether there is a correlation between diagnosis and DnaseX activity inthe serum. In total serum samples from patients with liver carcinoma(one patient), liver metastases originating from colorectal cancer (7patients), thyroid carcinoma (one patient), pancreatic carcinomas(patients), oesophageal carcinomas (4 patients), gastric carcinomas (17patients), colorectal carcinomas (22 patients), rectal carcinomas (8patients), colorectal adenomas (patients) and from 19 normal controlswere included.

To measure the DNase activity in sera from tumor patients we used aDNase activity test established for the determination of reduced DNaseactivity levels in systemic lupus erythematosus (SLE) patients. Usingthis test we were able to detect an enhanced DNase activity in sera oftumor patents. In the used DNase activity test the DNase present inserum leads to a degradation of the DNase substrate coated to the ELISAplate. Therefore the higher the DNase activity present in the serum thelower is the binding of the secondary antibody and the concomitant ELISAvalue (inverse relationship between signal height and DNase activity).

The results of the experiment are given in FIG. 7. Whereas sera of 8rectal carcinomas do not show elevated (or only weakly elevated) DNaseactivity levels, most sera of colon carcinoma and adenoma patents doshow elevated DNase activity levels. Also the pancreatic and the gastriccancer patents do show elevated DNase activity levels. Whereas a serumof a liver carcinoma patient does not show an elevated DNase activitylevel, all 7 liver metastasis sera do show extremely elevated DNaseactivity levels.

Immunohistochemical analysis demonstrated an overexpression of theDNaseX protein in more than 95% of bladder carcinomas. A DNase ac4vitytest with urine from bladder carcinoma patients revealed an enhancedDNase activity in patent's urine compared to urine from healthy controls(not shown).

In addition to the detection of elevated levels of DNase based on theenzymatic detection, we also performed standard ELISA experiments with acombination of capture and detection antibody directed against DNaseX. Acombination of a monoclonal antibody directed against DNaseX and apolyclonal antibody directed against DNaseX revealed the most specificresults in respect of sensitivity and specificity of the test Using thistest an enhanced level of DNaseX protein has been detected in bodyfluids like serum, sputum and urine of tumor patents (not shown).

Based on the overexpression of the DNaseX protein in tumors screeningtests can be efficiently performed which lead to the identification ofpatents with yet unrecognized tumors. Therefore the time point ofdetection is much earlier compared to standard tests like hemeoccult(fecal occult blood) test In addition tumor types can now be detectedwhich are until now extremely difficult to detect like gastric cancer orpancreas cancer. Since this cancer early detection test is able toidentify almost all or all tumor entities the test fulfills an importantcriteria for a general and cheap early cancer test for a mass screening.

1-26. (canceled)
 27. A method for diagnosis of carcinomas and theirprecursor lesions and/or prognosis of disease course comprising a)obtaining a cell containing tissue sample from an individual b)determining the level and/or subcellular localization of DNase-Xmolecules in the cells of said tissue; c) comparing the level and/orsubcellular localization of DNase-X molecules within said sample to thecontents within a corresponding control sample, not affected by thedisease being tested; d) wherein the diagnosis or prognosis of diseasecourse is predicted from considering a significant increased levelrelative to the wild type level of DNase-X molecule in said tissuesample and/or cellular nuclei as indicative of said disorder or of theprognosis of the disease course.
 28. The method according to claim 1,wherein the detection of the DNase-X molecules comprises the detectionof the accessibility of particular regions of the DNase-X molecules. 29.The method according to claim 1, wherein the sample is selected from agroup comprising blood, plasma, serum, liquor, lymph, bone marrow,swabs, washes, lavages, secretions, transsudates, exsudates, sputum,stool, urine, semen, cell- and tissue-samples, punctuates or biopsies.30. The method according to claim 1, wherein the carcinoma is selectedfrom a group comprising cancer of the head and the neck, cancer of therespiratory tract, cancer of the gastrointestinal tract, cancer of theskin and its appendages, cancer of the central and peripheral nervoussystem, cancer of the urinary system, cancer of the reproductive system,cancer of the endocrine system, cancer of the soft tissues and bone,cancer of the lymphopoietic and hematopoietic system, breast cancer,lung cancer, cervical cancer, colorectal cancer or anogenital cancer.31. The method according to claim 1, wherein the detection of the levelof the DNase-X molecule is carried out using at least one probespecifically binding to the marker molecules to be detected.
 32. Themethod according to claim 5, wherein the probe is detectably labelled.33. The method according to claim 6, wherein the label is selected fromthe group consisting of a radioisotope, a bioluminescent compound, achemiluminescent compound, a fluorescent compound, a metal chelate, abiologically relevant binding structure such as biotin or digoxygenin oran enzyme.
 34. The method according to claim 5, wherein at least oneprobe is an antibody, a fragment of an antibody, a peptidomimeticcomprising an antigen binding epitope or a mini-antibody.
 35. The methodaccording to claim 8, wherein the detection comprises animmuno-cytochemical detection procedure.
 36. The method according toclaim 5, wherein at least one probe being a nucleic acid hybridising toa marker nucleic acid is used for the detection of the DNase-X markermolecules.
 37. The method according to claim 10, wherein the detectionreaction comprises a nucleic acid amplification reaction.
 38. A probefor cancer, the probe comprising a probe specifically binding to orreacting with DNase-X and being capable of indicating amount and/orconcentration and/or localisation of DNase-X in a tissue sample or asample containing cells and/or cell fragments and/or cell nuclei.
 39. Amethod of identifying and obtaining a drug candidate for therapycarcinomas and their precursor lesions comprising the following steps:a) contacting a DNase-X polypeptide or a cell expressing saidpolypeptide in the presence of components capable of providing adetectable signal in response to DNase-X activity, cell proliferation orcell differentiation with said drug candidate to be screened underconditions to allow DNase-X activity, cell proliferation or changes incell differentiation and b) detecting presence or absence of a signal orincrease of the signal generated from DNase-X activity, cellproliferation or cell differentiation, wherein the presence or increaseof the signal is indicative for a putative drug.
 40. Kit for thedetection and/or treatment of carcinomas and their precursor lesions,comprising at least DNase-X or a compound selected from a groupcomprising a) a binding partner to a DNase-X polypeptide; b) anactivators/agonists or inhibitors/antagonists of a DNase-X polypeptide;c) an activator or inhibitor of the expression of a DNase-X polypeptide;and d) a drug candidate as described in claim
 13. 41. Pharmaceuticalcomposition useful for treating carcinomas and their precursor lesionscomprising at least DNase-X or a compound selected from a groupcomprising a) one or more DNase molecules being nucleic acids orpolypeptides; b) one or more activators/agonists orinhibitors/antagonists of a DNase polypeptide; c) one or more activatorsor inhibitors of the expression of a DNase polypeptide; d) one or morebinding partners of DNase polypeptides; and e) or one or more drugcandidates as described in claim 13; for production of a.
 42. Thepharmaceutical composition according to claim 15, wherein the carcinomais selected from a group comprising cancer of the head and the neck,cancer of the respiratory tract, cancer of the gastrointestinal tract,cancer of the skin and its appendages, cancer of the central andperipheral nervous system, cancer of the urinary system, cancer of thereproductive system, cancer of the endocrine system, cancer of the softtissues and bone, cancer of the lymphopoietic and hematopoietic system,breast cancer, anogenital cancer or colorectal cancer.